Peptides and combination of peptides for use in immunotherapy against CLL and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 62/265,615, filed 10 Dec. 2015, and Great Britain Application No. 1521746.6, filed 10 Dec. 2015, the content of each of these applications is herein incorporated by reference in their entirety.

This application also is related to PCT/EP2016/078718 filed 24 Nov. 2016, the content of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE (.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled “Sequence_Listing_2912919-058002_ST25.txt” created on 8 Dec. 2016, and 82,236 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to several novel peptide sequences and their variants derived from HLA class I molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses, or as targets for the development of pharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with morphologically mature, immunologically not completely matured B lymphocytes. CLL is diagnosed when at least 5000 B lymphocytes/μl are present in peripheral blood, whereof up to 55% are immature. Abnormal B cells show a characteristic phenotype expressing CD19, dim CD20, dim CD5, CD23, CD79a and dim IgM or dim IgD (Gribben, 2010).

Two different staging systems are used to classify CLL: The Rai system and the Binet system. The Rai staging system comprises five stages (0-IV) that classify the disease according to the progressive accumulation of abnormal cells. The Binet system uses three stages (A, B, C) to rank the disease according to the number of involved sites (http://www.cancer.gov/cancertopics/pdq/treatment/CLL/Patient).

CLL is primarily a disease of the elderly. The mean age at diagnosis is 72 years for sporadic cases and 58 years for familial cases. The incidence of CLL is higher in males than in females, with a male:female ratio of about 2:1 (Cartwright et al., 2002).

CLL is the most common leukemia in the Western world where it comprises about ⅓rd of all leukemias. Incidence rates are similar in the US and Europe, and estimated new cases are about 16,000 per year. CLL is more common in Caucasians than in Africans, rarer in Hispanics and Native Americans and seldom in Asians. In people of Asian origin, CLL incidence rates are 3-fold lower than in Caucasians (Gunawardana et al., 2008).

The five-year overall survival for patients with CLL is about 79% (http://www.cancer.net/cancer-types/leukemia-chronic-lymphocytic-cll/statistics). The prognosis for individual patients depends on the stage at the time of diagnosis and the occurrence of several prognostic factors. Following the Rai system, patients with stage 0 show a median survival of 10 years or more, while patients at stage I/II have a median survival of 7 years and patients at stage III/IV 0.75-4 years (Gribben, 2010). Several factors are associated with poor prognosis in CLL. These include high expression levels of ZAP-70 or CD38, an IgVH unmutated status and the cytogenetic aberrations del 17p or del 11q (Gribben, 2010).

While CLL is not curable at present, many patients show only slow progression of the disease or worsening of symptoms. As patients do not benefit from an early onset of treatment, the initial approach is “watch and wait” (Richards et al., 1999). For patients with symptomatic or rapidly progressing disease, several treatment options are available. These include chemotherapy, targeted therapy, immune-based therapies like monoclonal antibodies, CARs and active immunotherapy, and stem cell transplants.

Chemotherapeutic drugs used for CLL treatment are mostly alkylating agents like chlorambucil and cyclophosphamide or purine analogues like fludarabine. The German CLL Study Group (GCLLSG) CCL4 demonstrated that fludarabine/cyclophosphamide combinational therapy is superior to sole fludarabine treatment (complete remission (CR) of 24% vs. 7%) (Eichhorst et al., 2006).

Ibrutinib and idelalisib are kinase inhibitors that target molecules in the B-cell receptor signaling cascade. Ibrutinib inhibits Bruton's tyrosine kinase (BTK), a src-related cytoplasmic tyrosine kinase important for B-cell maturation, and is used for initial or second-line therapy (Byrd et al., 2013; O'Brien et al., 2014). Idelalisib is a PI3K-delta inhibitor used in combination with rituximab in refractory CLL (Furman et al., 2014).

Hematopoietic stem cell transplants (HSCTs) can be considered for patients with poor prognosis, e.g. patients with del 17p or p53 mutations. HSCTs can either be allogeneic, where the transplanted cells are donated from an HLA-matched person, or autologous, where the patients' own stem cells are re-infused after chemotherapy (Schetelig et al., 2008).

Monoclonal antibodies are widely used in hematologic malignancies. This is due to the knowledge of suitable antigens based on the good characterization of immune cell surface molecules and the accessibility of tumor cells in blood or bone marrow. Common monoclonal antibodies used in CLL therapy target either CD20 or CD52. Rituximab, the first monoclonal anti-CD20 antibody originally approved by the FDA for treatment of NHLs, is now widely used in CLL therapy. Combinational treatment with rituximab/fludarabine/cyclophosphamide leads to higher CR rates and improved overall survival (OS) than the combination fludarabine/cyclophosphamide and has become the preferred treatment option (GCLLSG CLL8) (Hallek et al., 2008). Ofatumomab targets CD20 and is used for therapy of refractory CLL patients (Wierda et al., 2011). Obinutuzumab is another monoclonal anti-CD20 antibody used in first-line treatment in combination with chlorambucil (Goede et al., 2014).

Alemtuzumab is an anti-CD52 antibody used for treatment of patients with chemotherapy-resistant disease or patients with poor prognostic factors as del 17p or p53 mutations (Parikh et al., 2011). Novel monoclonal antibodies target CD37 (otlertuzumab, BI 836826, IMGN529 and (177)Lu-tetulomab) or CD40 (dacetuzumab and lucatumumab) and are tested in pre-clinical settings (Robak and Robak, 2014).

Several completed and ongoing trials are based on engineered autologous chimeric antigen receptor (CAR)-modified T cells with CD19 specificity (Maus et al., 2014). So far, only the minority of patients showed detectable or persistent CARs. One partial response (PR) and two complete responses (CR) have been detected in the CAR T-cell trials by Porter et al. and Kalos et al. (Kalos et al., 2011; Porter et al., 2011).

Active immunotherapy includes the following strategies: gene therapy, whole modified tumor cell vaccines, DC-based vaccines and TAA-derived peptide vaccines.

Approaches in gene therapy make use of autologous genetically modified tumor cells. These B-CLL cells are transfected with immuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha, GM-CSF, CD80, CD40L, LFA-3 and ICAM-1 to improve antigen presentation and T cell activation (Carballido et al., 2012). While specific T-cell responses and reduction in tumor cells are readily observed, immune responses are only transient.

Several studies have used autologous DCs as antigen presenting cells to elicit anti-tumor responses. DCs have been loaded ex vivo with tumor associated peptides, whole tumor cell lysate, tumor-derived RNA or DNA. Another strategy uses whole tumor cells for fusion with DCs and generation of DC-B-CLL-cell hybrids. Transfected DCs initiated both CD4+ and CD8+ T-cell responses (Muller et al., 2004). Fusion hybrids and DCs loaded with tumor cell lysate or apoptotic bodies increased tumor-specific CD8+ T-cell responses. Patients that showed a clinical response had increased IL-12 serum levels and reduced numbers of Tregs (Palma et al., 2008).

Different approaches use altered tumor cells to initiate or increase CLL-specific immune responses. An example for this strategy is the generation of trioma cells: B-CLL cells are fused to anti-Fc receptor expressing hybridoma cells that have anti-APC specificity. Trioma cells induced CLL-specific T-cell responses in vitro (Kronenberger et al., 2008). Another strategy makes use of irradiated autologous CLL cells with Bacillus Calmette-Guerin as an adjuvant as a vaccine. Several patients showed a reduction in leukocyte levels or stable disease (Hus et al., 2008). Besides isolated CLL cells, whole blood from CLL patients has been used as a vaccine after preparation in a blood treatment unit. The vaccine elicited CLL-specific T-cell responses and led to partial clinical responses or stable disease in several patients (Spaner et al., 2005).

Several TAAs are overexpressed in CLL and are suitable for vaccinations. These include fibromodulin (Mayr et al., 2005), RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT (Counter et al., 1995), the oncofetal antigen-immature laminin receptor protein (OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al., 2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt et al., 2002) and the tumor-derived IgVHCDR3 region (Harig et al., 2001; Carballido et al., 2012).

A phase I clinical trial was conducted using the RHAMM-derived R3 peptide as a vaccine. 5 of 6 patients had detectable R3-specific CD8+ T-cell responses (Giannopoulos et al., 2010).

Considering the severe side-effects and expense associated with treating cancer, there is a need to identify factors that can be used in the treatment of cancer in general and CLL in particular. There is also a need to identify factors representing biomarkers for cancer in general and CLL in particular, leading to better diagnosis of cancer, assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting of cancer cells while minimizing side effects. Cancer immunotherapy makes use of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprises the following major groups:

-   a) Cancer-testis antigens: The first TAAs ever identified that can     be recognized by T cells belong to this class, which was originally     called cancer-testis (CT) antigens because of the expression of its     members in histologically different human tumors and, among normal     tissues, only in spermatocytes/spermatogonia of testis and,     occasionally, in placenta. Since the cells of testis do not express     class I and II HLA molecules, these antigens cannot be recognized by     T cells in normal tissues and can therefore be considered as     immunologically tumor-specific. Well-known examples for CT antigens     are the MAGE family members and NY-ESO-1. -   b) Differentiation antigens: These TAAs are shared between tumors     and the normal tissue from which the tumor arose. Most of the known     differentiation antigens are found in melanomas and normal     melanocytes. Many of these melanocyte lineage-related proteins are     involved in biosynthesis of melanin and are therefore not tumor     specific but nevertheless are widely used for cancer immunotherapy.     Examples include, but are not limited to, tyrosinase and     Melan-A/MART-1 for melanoma or PSA for prostate cancer. -   c) Over-expressed TAAs: Genes encoding widely expressed TAAs have     been detected in histologically different types of tumors as well as     in many normal tissues, generally with lower expression levels. It     is possible that many of the epitopes processed and potentially     presented by normal tissues are below the threshold level for T-cell     recognition, while their over-expression in tumor cells can trigger     an anticancer response by breaking previously established tolerance.     Prominent examples for this class of TAAs are Her-2/neu, survivin,     telomerase, or WT1. -   d) Tumor-specific antigens: These unique TAAs arise from mutations     of normal genes (such as β-catenin, CDK4, etc.). Some of these     molecular changes are associated with neoplastic transformation     and/or progression. Tumor-specific antigens are generally able to     induce strong immune responses without bearing the risk for     autoimmune reactions against normal tissues. On the other hand,     these TAAs are in most cases only relevant to the exact tumor on     which they were identified and are usually not shared between many     individual tumors. Tumor-specificity (or -association) of a peptide     may also arise if the peptide originates from a tumor- (-associated)     exon in case of proteins with tumor-specific (-associated) isoforms. -   e) TAAs arising from abnormal post-translational modifications: Such     TAAs may arise from proteins which are neither specific nor     overexpressed in tumors but nevertheless become tumor associated by     posttranslational processes primarily active in tumors. Examples for     this class arise from altered glycosylation patterns leading to     novel epitopes in tumors as for MUC1 or events like protein splicing     during degradation which may or may not be tumor specific. -   f) Oncoviral proteins: These TAAs are viral proteins that may play a     critical role in the oncogenic process and, because they are foreign     (not of human origin), they can evoke a T-cell response. Examples of     such proteins are the human papilloma type 16 virus proteins, E6 and     E7, which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by molecules of the major histocompatibility complex (MHC). The antigens that are recognized by the tumor specific T lymphocytes, that is, the epitopes thereof, can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc. which are expressed and, as compared to unaltered cells of the same origin, usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II. MHC class I molecules are composed of an alpha heavy chain and beta-2-microglobulin, MHC class II molecules of an alpha and a beta chain. Their three-dimensional conformation results in a binding groove, which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They present peptides that result from proteolytic cleavage of predominantly endogenous proteins, defective ribosomal products (DRIPs) and larger peptides. However, peptides derived from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules. This non-classical way of class I presentation is referred to as cross-presentation in the literature (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules can be found predominantly on professional antigen presenting cells (APCs), and primarily present peptides of exogenous or transmembrane proteins that are taken up by APCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive T cells bearing the appropriate T-cell receptor (TCR), whereas complexes of peptide and MHC class II molecules are recognized by CD4-positive-helper-T cells bearing the appropriate TCR. It is well known that the TCR, the peptide and the MHC are thereby present in a stoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing and sustaining effective responses by CD8-positive cytotoxic T cells. The identification of CD4-positive T-cell epitopes derived from tumor associated antigens (TAA) is of great importance for the development of pharmaceutical products for triggering anti-tumor immune responses (Gnjatic et al., 2003). At the tumor site, T helper cells, support a cytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006) and attract effector cells, e.g. CTLs, natural killer (NK) cells, macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system, especially professional antigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells, macrophages, dendritic cells. In cancer patients, cells of the tumor have been found to express MHC class II molecules (Dengjel et al., 2006).

Elongated (longer) peptides of the invention can act as MHC class II active epitopes.

T-helper cells, activated by MHC class II epitopes, play an important role in orchestrating the effector function of CTLs in anti-tumor immunity. T-helper cell epitopes that trigger a T-helper cell response of the TH1 type support effector functions of CD8-positive killer T cells, which include cytotoxic functions directed against tumor cells displaying tumor-associated peptide/MHC complexes on their cell surfaces. In this way tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in the absence of CD8-positive T lymphocytes, CD4-positive T cells are sufficient for inhibiting manifestation of tumors via inhibition of angiogenesis by secretion of interferon-gamma (IFNγ) (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cells as direct anti-tumor effectors (Braumuller et al., 2013; Tran et al., 2014).

Since the constitutive expression of HLA class II molecules is usually limited to immune cells, the possibility of isolating class II peptides directly from primary tumors was previously not considered possible. However, Dengjel et al. were successful in identifying a number of MHC Class II epitopes directly from tumors (WO 2007/028574, EP 1 760 088 B1).

Since both types of response, CD8 and CD4 dependent, contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens recognized by either CD8+ T cells (ligand: MHC class I molecule+peptide epitope) or by CD4-positive T-helper cells (ligand: MHC class II molecule+peptide epitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immune response, it also must bind to an MHC-molecule. This process is dependent on the allele of the MHC-molecule and specific polymorphisms of the amino acid sequence of the peptide. MHC-class-I-binding peptides are usually 8-12 amino acid residues in length and usually contain two conserved residues (“anchors”) in their sequence that interact with the corresponding binding groove of the MHC-molecule. In this way, each MHC allele has a “binding motif” determining which peptides can bind specifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T cells bearing specific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or -associated antigens, and to be used in a therapy, particular prerequisites must be fulfilled. The antigen should be expressed mainly by tumor cells and not, or in comparably small amounts, by normal healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells as compared to normal healthy tissues. It is furthermore desirable that the respective antigen is not only present in a type of tumor, but also in high concentrations (i.e. copy numbers of the respective peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins directly involved in transformation of a normal cell to a tumor cell due to their function, e.g. in cell cycle control or suppression of apoptosis. Additionally, downstream targets of the proteins directly causative for a transformation may be up-regulated and thus may be indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets of a vaccination approach (Singh-Jasuja et al., 2004). It is essential that epitopes are present in the amino acid sequence of the antigen, in order to ensure that such a peptide (“immunogenic peptide”), being derived from a tumor associated antigen, leads to an in vitro or in vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as a T-cell epitope. A prerequisite for the induction of an in vitro or in vivo T-cell-response is the presence of a T cell having a corresponding TCR and the absence of immunological tolerance for this particular epitope.

Therefore, TAAs are a starting point for the development of a T cell based therapy including but not limited to tumor vaccines. The methods for identifying and characterizing the TAAs are usually based on the use of T-cells that can be isolated from patients or healthy subjects, or they are based on the generation of differential transcription profiles or differential peptide expression patterns between tumors and normal tissues. However, the identification of genes over-expressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide precise information as to the use of the antigens being transcribed from these genes in an immune therapy. This is because only an individual subpopulation of epitopes of these antigens are suitable for such an application since a T cell with a corresponding TCR has to be present and the immunological tolerance for this particular epitope needs to be absent or minimal. In a very preferred embodiment of the invention it is therefore important to select only those over- or selectively presented peptides against which a functional and/or a proliferating T cell can be found. Such a functional T cell is defined as a T cell, which upon stimulation with a specific antigen can be clonally expanded and is able to execute effector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs) and antibodies or other binding molecules (scaffolds) according to the invention, the immunogenicity of the underlying peptides is secondary. In these cases, the presentation is the determining factor.

SUMMARY

In a first aspect of the present invention, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant sequence thereof which is at least 77%, preferably at least 88%, homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 385, wherein said variant binds to MHC and/or induces T cells cross-reacting with said peptide, or a pharmaceutical acceptable salt thereof, wherein said peptide is not the underlying full-length polypeptide.

The present invention further relates to a peptide of the present invention comprising a sequence that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof, which is at least 77%, preferably at least 88%, homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 385, wherein said peptide or variant thereof has an overall length of between 8 and 100, preferably between 8 and 30, and most preferred of between 8 and 14 amino acids.

The following tables show the peptides according to the present invention, their respective SEQ ID NOs, and the prospective source (underlying) genes for these peptides. All peptides in Table 1 and Table 2 bind to HLA-A*02. The peptides in Table 2 have been disclosed before in large listings as results of high-throughput screenings with high error rates or calculated using algorithms, but have not been associated with cancer at all before. The peptides in Table 3 are additional peptides that may be useful in combination with the other peptides of the invention. The peptides in Table 4 are furthermore useful in the diagnosis and/or treatment of various other malignancies that involve an over-expression or over-presentation of the respective underlying polypeptide.

TABLE 1 Peptides according to the present invention. SEQ ID Official Gene No. Sequence GeneID(s) Symbol(s) 1 AIPPSFASIFL      3507 IGHM 2 ALHRPDVYL      3507 IGHM 3 VIAELPPKV      3507 IGHM 4 VIAELPPKVSV      3507 IGHM 5 ALIFKIASA      4214 MAP3K1 6 ALDTLEDDMTI      2222 FDFT1 7 ALLERTGYTL     10236, HNRNPR, SYNCRIP     10492 8 ALAASALPALV      6124 RPL4 9 ALCDTLITV     27033 ZBTB32 10 FVYGESVEL     27033 ZBTB32 11 ALFTFJPLTV     54665 RSBN1 12 ALGEDEITL     25909, AHCTF1, AHCTF1P1    285116 13 VVDGMPPGV      2969, GTF2I, GTF2IP1      2970 14 ALLRLLPGL     25920 COBRA1 15 ALPEVSVEA     79649 MAP7D3 16 ALPGGAAVAAV     79697 C14orf169 17 ALTKTNLQL      9750 FAM65B 18 LLGEFSIKM      9750 FAM65B 19 QVMEKLAAV      9750 FAM65B 20 ALVDPGPDFVV       546 ATRX 21 ALWAGLLTL      2208 FCER2 22 ALWDPVIEL     27340 UTP20 23 ALYLTEVFL     55024 BANK1 24 AMAGDVVYA    160365 CLECL1 25 ATYSGLESQSV      3185 HNRNPF 26 AVLLVLPLV     11322 TMC6 27 AVLGLVWLL     11322 TMC6 28 AVLHLLLSV      1535 CYBA 29 AVLQAVTAV     58155 PTBP2 30 ELLEGSEIYL    147007, TMEM199, SARM1     23098 31 ELMHGVAGV    165631 PARP15 32 FIDKFTPPV      3117, HLA-DQA1, HLA-DRA      3122 33 FIINSSNIFL    253959 RALGAPA1 34 YLPYIFPNI    253959 RALGAPA1 35 FILPSSLYL     81539 SLC38A1 36 FILTHVDQL    257106 ARHGAP30 37 FIMEGGAMVL      4174 MCM5 38 FIMEGGAMV      4174 MCM5 39 FLDALLTDV     79915 ATAD5 40 FLDEDDMSL     23141 ANKLE2 41 FLDPSLDPLL     58508, MLL3, BAGE3     85318 42 FLEEGGVVTV      5810 RAD1 43 FLLGPEALSFA     55183 RIF1 44 FLLSINDFL    157680 VPS13B 45 FLPELPADLEA     11064 CNTRL 46 YIIDSAQAV     11064 CNTRL 47 FLSDQPEPYL    160518, DENND5B, DENND5A     23258 48 FLSPQQPPLL      8621 CDK13 49 FLTDLFAQL     55671 SMEK1 50 FLFEPVVKAFL     55671 SMEK1 51 FLVEAPHDWDL      9790 BMS1 52 FLVETGFLHV 100268168, WDR31, MYB    114987,      4602 53 FLWQHVELVL     55181 SMG8 54 FLYPFPLALF    440026 TMEM41B 55 FMEPTLLML        23 ABCF1 56 FVFEAPYTL    139818 DOCK11 57 GLSEISLRL    139818 DOCK11 58 YIQQGIFSV    139818 DOCK11 59 FVFGDENGTVSL     79084 WDR77 60 FVLDHEDGLNL      4926 NUMA1 61 FVYFIVREV    285527 FRYL 62 GIIDGSPRL     80183 KIAA0226L 63 SLAHVAGCEL     80183 KIAA0226L 64 KLLESVASA     80183 KIAA0226L 65 GLDDMKANL      5079 PAX5 66 SLAGGLDDMKA      5079 PAX5 67 GLDDVTVEV     51019 CCDC53 68 GLDQQFAGLDL      1654 DDX3X 69 GLHQREIFL      9922 IQSEC1 70 FVPDTPVGV      9922 IQSEC1 71 GLKHDIARV    388512 CLEC17A 72 GLLDAGKMYV       911 CD1C 73 GLLEVISALQL      9816 URB2 74 GLLRASFLL     11344, TWF2, TLR9     54106 75 GLSIFAQDLRL     11344, TWF2, TLR9     54106 76 GLLRIIPYL     23352 UBR4 77 GLLRLTWFL     11214 AKAP13 78 GLPSFLTEV 100532732, MSH5-SAPCD1, MSH5      4439 79 GLQAKIQEA     23224 SYNE2 80 VLIEDELEEL     23224 SYNE2 81 WLVGQEFEL     23224 SYNE2 82 GLQSGVDIGV      1265 CNN2 83 GQGEVLVYV      2316 FLNA 84 GVMDVNTAL    391370, RPS12P4, RPS12      6206 85 HLMLHTAAL     80012 PHC3 86 HLYPGAVTI     55103 RALGPS2 87 HQIEAVDGEEL     10567 RABAC1 88 ILDEIGADVQA     29920 PYCR2 89 ILDFGTFQL     54832 VPS13C 90 VIADLGLIRV     54832 VPS13C 91 ILDLNTYNV      5336 PLCG2 92 ILEPLNPLL      9330 GTF3C3 93 ILFNTQINI     64801 ARV1 94 ILFPLRFTL      9675 TTI1 95 ILGYMAHEHKV      6530 SLC6A2 96 ILIDKTSFV       951 CD37 97 LLFATQITL       951 CD37 98 SLIKYFLFV       951 CD37 99 ILIFHSVAL     84636 GPR174 100 ILNNEVFAI     26999 CYFIP2 101 ILVVIEPLL     23451 SF3B1 102 IQDRAVPSL      3930 LBR 103 KLGGTPAPA      5218 CDK14 104 KLILLDTPLFL    253190, SERHL2, SERHL     94009 105 KLMNDIADI       472 ATM 106 FMASHLDYL       472 ATM 107 ILYNLYDLL       472 ATM 108 VIYTLIHYI       472 ATM 109 KLWEGLTELV     10235 RASGRP2 110 LLFDHLEPMEL     10235 RASGRP2 111 KLWNVAAPLYL     92105 INTS4 112 KTLDVDATYEI      3978 LIG1 113 KVPAEEVLVAV     91782 CHMP7 114 LIPEGPPQV     64224 HERPUD2 115 LLFDKLYLL     25885 POLR1A 116 LLIGATMQV     23225 NUP210 117 LLILENILL     23225 NUP210 118 RLLILENILL     23225 NUP210 119 VLPAEFFEV     23225 NUP210 120 AIDAALTSV     23225 NUP210 121 RLYEITIEV     23225 NUP210 122 LLIPVVPGV     55178 RNMTL1 123 LLLAEAELLTL     57724 EPG5 124 LLLEETEKQAV     92154, MTSS1L, MTSS1      9788 125 LLLEIGEVGKLFV     23158 TBC1D9 126 LLPEGGITAI      9904 RBM19 127 LLPTAPTTV     55729 ATF7IP 128 LLSEEEYHL      9793 CKAP5 129 LLVGTLDVV     23061, TBC1D9B, TBC1D9     23158 130 LLVLIPVYL     23344 ESYT1 131 LQALEVLKI     27304 MOCS3 132 LVYEAIIMV     27250 PDCD4 133 YLLSGDISEA     27250 PDCD4 134 MLLEHGITLV    200576 PIKFYVE 135 MTAGFSTIAGSV     64078 SLC28A3 136 NLDKLWTLV    389435, RPL27AP6, RPL27A      6157 137 NLIKTVIKL     25816 TNFAIP8 138 NLLDIDAPVTV      5530 PPP3CA 139 NLTDVVEKL    120892 LRRK2 140 QIAELPATSV    401145 CCSER1 141 QILSEIVEA     10724 MGEA5 142 QLDEPAPQV    387032, ZKSCAN4, ZKSCAN3     80317 143 QLLDTYFTL      7405 UVRAG 144 QLPPFPREL      8498 RANBP3 145 SALDTITTV      3832 KIF11 146 SIIEGPIIKL     23347 SMCHD1 147 SILETVATL    253714 MMS22L 148 SIVASLITV    221188 GPR114 149 SLDNGGYYI      4067 LYN 150 SLFDQPLSII    117289 TAGAP 151 SLFDSAYGA      2313 FLI1 152 SLIRILQTI    160518 DENND5B 153 SLLAELHVL    115352 FCRL3 154 SLLAELHVLTV    115352 FCRL3 155 SLMLEVPAL      1756 DMD 156 SLNIGDVQL       973 CD79A 157 SLNIRDFTM    147694, ZNF548, ZNF17      7565 158 SLPEAPLDV     29123 ANKRD11 159 SLQEEKLIYV     84668 FAM126A 160 SLSFLVPSL      9631 NUP155 161 SMDDGMINV      7011 TEP1 162 SMKDDLENV      3688 ITGB1 163 SQLDISEPYKV      3394 IRF8 164 SVHKGFAFV 101060301, HNRNPC, HNRNPCL1      3183,    343069,    440563,    649330 165 TLDDDLDTV     51092 SIDT2 166 TLDPNQVSL     85464 SSH2 167 TLDTSKLYV      5923 RASGRF1 168 YLLDQSFVM      5923 RASGRF1 169 TLLLGLTEV     55423 SIRPG 170 TLTFRVETV     51379 CRLF3 171 TLVPPAALISI      6907 TBL1X 172 TLYDMLASI     10973 ASCC3 173 TVIENIHTI     23358 USP24 174 VLAELPIIVV      8295 TRRAP 175 FTVPRVVAV      8295 TRRAP 176 VLAEQNIIPSA     54855 FAM46C 177 VLDDRELLL     23178 PASK 178 VLFFNVQEV     57536 KIAA1328 179 VLLGLEMTL      1606 DGKA 180 LLKDGPEIGL      1606 DGKA 181 VLLSIPFVSV     94103 ORMDL3 182 VLLSVPGPPV      2264 FGFR4 183 VLMPTVYQQGV     54472 TOLLIP 184 VLSHNLYTV     50619 DEF6 185 VMDDQRDLI       972 CD74 186 VMDPTKILI    255967 PAN3 187 VMDTHLVNI      9779 TBC1D5 188 VMGDIPAAV      4678 NASP 189 VMLEMTPEL      3112, HLA-DOB, TAP2      6891 190 VVMGTVPRL      1729 DIAPH1 191 YIFDGSDGGV      5287 PIK3C2B 192 YIQEYLTLL     57674 RNF213 193 YLDLSNNRL     81793 TLR10 194 YLDNVLAEL      1840, DTX1, DTX4     23220 195 YLGGFALSV      6850 SYK 196 YLLLQTYVL     51259 TMEM216 197 YLQEVPILTL      1633 DCK 198 YLTFLPAEV     64135 IFIH1 199 YLVELSSLL     80279 CDK5RAP3 200 YMFEEVPIVI      8667 EIF3H 201 YQLELHGIEL      5698 PSMB9 202 YVDDVFLRV     10347 ABCA7 203 ALLSSQLAL      8925 HERC1 204 GLLQINDKIAL      5000 ORC4 205 GLSQANFTL     10288, LILRB2, LILRB1,     10859, LILRB5, LILRA1,     10990, LILRB3, LILRA3,     11024, LILRA2, LILRA4,     11025, LILRP2, LILRA6     11026,     11027,     23547,     79166,     79168 206 HMQDVRVLL     84824 FCRLA 207 IIADLDTTIMFA      7094, TLN1, TLN2     83660 208 ILLKTEGINL    166379 BBS12 209 ILQAELPSL     57508 INTS2 210 KLLVQDFFL      8564 KMO 211 LIDVKPLGV    134510 UBLCP1 212 NIIEAINELLV      8892 EIF2B2 213 RLLYQLVFL      3566 IL4R 214 RLQELTEKL    255394 TCP11L2 215 VMQDIVYKL     84759 PCGF1 216 WLAGDVPAA     24148 PRPF6 217 ALDEPPYLTV     51742 ARID4B 218 ALGEEWKGYVV      6194, RPS6    728131 219 ALLNLLESA      5788 PTPRC 220 ALPEILFAKV       643 CXCR5 221 ALVSTIIMV     80228 ORAI2 222 ALWELSLKI      2889 RAPGEF1 223 ALWVSQPPEI    259197 NCR3 224 AMEALVVEV      1660 DHX9 225 ILQERELLPV      1660 DHX9 226 AMNISVPQV     23387 SIK3 227 FLAEASVMTQL      1445 CSK 228 FLGGLSPGV      2968 GTF2H4 229 FLLNLQNCHL     55608 ANKRD10 230 FLQDSKVIFV      4772 NFATC1 231 FLYIRQLAI     26155, NOC2L    401010 232 FMHQIIDQV     79789 CLMN 233 GIIDINVRL     51163 DBR1 234 GLDDAEYAL      7376 NR1H2 235 GLDDLLLFL      8914 TIMELESS 236 GLLESGRHYL      9744 ACAP1 237 GLQENLDVVV      8881 CDC16 238 GLVETELQL     55690 PACS1 239 ILAGEMLSV      7203 CCT3 240 ILARDILEI      5714 PSMD8 241 ILGDILLKV     79831 KDM8 242 ILLGIQELL      7329 UBE2I 243 ILPTLEKELFL     79886 CAAP1 244 ILQALAVHL     54936 ADPRHL2 245 KIMDYSLLLGV     23396 PIP5K1C 246 KLDETGVAL      7155 TOP2B 247 KLKDRLPSI     81606 LBH 248 KTVEPPISQV     57466 SCAF4 249 LLPTGVFQV     51131 PHF11 250 LLVQEPDGLMV    404734, ANKHD1-EIF4EBP3,     54882 ANKHD1 251 LLYDNVPGA     11262 SP140 252 NLLDPGSSYLL      5074 PAWR 253 NLWSVDGEVTV     92017 SNX29 254 QLIPKLIFL     57705 WDFY4 255 YLFEEAISM     57705 WDFY4 256 QLLPVSNVVSV     55374 TMCO6 257 RIINGIIISV     84329 HVCN1 258 RLDYITAEI     10471 PFDN6 259 RLLDEQFAVL      9026 HIP1R 260 SLDDVEGMSV     80196 RNF34 261 SLVEAQGWLV    161176 SYNE3 262 SLWNAGTSV      3495 IGHD 263 SQWEDIHVV     55843 ARHGAP15 264 TILDYINVV     89790, SIGLEC10, SIGLEC12     89858 265 TLLADDLEIKL      2124 EVI2B 266 TLLDQLDTQL      8678 BECN1 267 TLLDWQDSL      8289 ARID1A 268 TLLQVFHLL     63892 THADA 269 TLTDEQFLV       738 VPS51 270 TVLPVPPLSV      5430 POLR2A 271 VIRNIVEAA 100128168, RPS26P39, RPS26P32, 100996747,  RPS26P11, RPS26,    389472,  RPS26P28, RPS26P25,    441502, RPS26P58      6231,    643003,    728937,    729188 272 VLDELPPLI     55832, CAND1, C22orf32     91689 273 VLGEYSYLL     23431 AP4E1 274 VLLEYHIAYL      8087 FXR1 275 VLLFIEHSV    134265, AFAP1L1, LAPTM5      7805 276 VLNDGAPNV    117246 FTSJ3 277 VMILKLPFL     25929 GEMIN5 278 YLDDLLPKL     89910 UBE3B 279 YMAPEVVEA      2872 MKNK2 280 YTLDSLYWSV     81887 LAS1L J = phospho-serine

KIAA0226L (also known as C13orf18) encodes KIAA0226-like and is located on chromosome 13q14.13 (RefSeq, 2002). KIAA0226L is thought to be a tumor suppressor gene and is hyper-methylated in cervical cancer. Re-activation of KIAA0226L leads to decreased cell growth, viability, and colony formation (Huisman et al., 2015; Eijsink et al., 2012; Huisman et al., 2013). The methylation pattern of KIAA0226L can be used to differ between precursor lesions and normal cervix cancer (Milutin et al., 2015). The methylation pattern of KIAA0226L cannot be used as specific biomarker for cervical cancer (Sohrabi et al., 2014). Re-activation of KIAA0226L partially de-methylates its promotor region and also decreases repressive histone methylations (Huisman et al., 2013).

FCRL3, also known as FCRH3 or IRTA3, encodes Fc receptor like 3, one of several FC receptor-like glycoproteins of the immunoglobulin receptor superfamily which may play a role in regulation of the immune system (RefSeq, 2002). FCRL3 is up-regulated on CD4+CD26− T cells in Sézary syndrome patients with a high tumor burden, suggesting that FCRL3 expression correlates with a high circulating tumor burden (Wysocka et al., 2014). FCRL3 is up-regulated in chronic lymphocytic leukemia (Polson et al., 2006). FCRL3 is a potential marker with prognostic relevance in chronic lymphocytic leukemia (Zucchetto et al., 2011)

TABLE 2 Additional peptides according to the present invention with no prior known cancer association. SEQ ID Official Gene No. Sequence GeneID(s) Symbol(s) 281 NLLDDRGMTAL    146206 RLTPR 282 LLRDGIELV    152926 PPM1K 283 ILQPMDIHV     54832 VPS13C 284 LLSAAEPVPA       974 CD79B 285 GVATAGCVNEV      3394 IRF8 286 FLLEDLSQKL      2177 FANCD2 287 FLWEEKFNSL     10750 GRAP 288 GLAESTGLLAV     50855, PARD6A, PARD6G     84552 289 ILEEQPMDMLL     84301 DDI2 290 LANPHELSL     84301 DDI2 291 ILLNEDDLVTI     57724 EPG5 292 AAALIIHHV     25942 SIN3A 293 ALDIMIPMV      4217 MAP3K5 294 ALLDQLHTLL      8202 NCOA3 295 ALLQKLQQL    122618 PLD4 296 FIAPTGHSL     23157, SEPT6, SEPT11     55752 297 FLVEPQEDTRL      8237 USP11 298 IILPVEVEV     23505 TMEM131 299 ILEENIPVL     55653 BCAS4 300 ILLNPAYDVYL     23225 NUP210 301 AASPIITLV     23225 NUP210 302 ILQDLTFVHL      2889 RAPGEF1 303 ILSQPTPSL     29028 ATAD2 304 LAIVPVNTL     23132 RAD54L2 305 LLFPQIEGIKI     26263 FBXO22 306 VVAEELENV     26263 FBXO22 307 LLLTKPTEA      5336 PLCG2 308 SLYDVSRMYV      5336 PLCG2 309 ILYGTQFVL      5336 PLCG2 310 LLSTLHLLV     64098 PARVG 311 LLVDVEPKV 100129492, SNRPD1      6632 312 LLYNSTDPTL     64005 MYO1G 313 LMADLEGLHL      8546 AP3B1 314 LMKDCEAEV      5518 PPP2R1A 315 LVYEAPETV     57448 BIRC6 316 MLLEHGITL    200576 PIKFYVE 317 NLLAHIWAL     58513 EPS15L1 318 NLQVTQPTV 100505503, RPS17L, RPS17P16,    402057, RPS17P5, RPS17    442216,      6218 319 QVIPQLQTV      6671 SP4 320 TIAPVTVAV      6671 SP4 321 RLLEFELAQL      6093 ROCK1 322 SLASIHVPL     23163 GGA3 323 SLDLFNCEV     10541, ANP32B, ANP32C,      23520, ANP32BP1, ANP32AP1,    646791, ANP32A    647020,    723972,      8125 324 SLYSALQQA     11190 CEP250 325 TLENGVPCV     23240 KIAA0922 326 VLAFLVHEL     11176 BAZ2A 327 VLIKWFPEV     54509 RHOF 328 VLLPQETAEIHL      5292 PIM1 329 VLMDGSVKL     11184 MAP4K1 330 VLMWEIYSL       695 BTK 331 VLWELAHLPTL     23358 USP24 332 VMIQHVENL      4033 LRMP 333 VTLEFPQLIRV    126382 NR2C2AP 334 YLLEEKIASL     51199 NIN 335 YLYQEQYFI     10668 CGRRF1 336 YMAVTTQEV     79840 NHEJ1 337 YMYEKESEL     22806 IKZF3 338 FLDMTNWNL     64771 C6orf106 339 GLWGTVVNI     25914 RTTN 340 KLLEEICNL     10075 HUWE1 341 LLAELPASVHA      2319 FLOT2 342 SLITPLQAV      5591 PRKDC 343 TLLEALDCI    158078, EEF1A1P5, EEF1A1      1915 344 VLAFENPQV     25852 ARMC8 345 YLIEPDVELQRI     25852 ARMC8 346 VLVQVSPSL     23404 EXOSC2 347 YLGPVSPSL      4154 MBNL1 348 ALAKPPVVSV     57634 EP400 349 ALATHILSL     11215 AKAP11 350 ALEDRVWEL    285025 CCDC141 351 ALSEKLARL    115106 HAUS1 352 ALVFELHYV     57705 WDFY4 353 ATPMPTPSV     55206 SBNO1 354 FIMDDPAGNSYL      8882 ZNF259 355 FIWPMLIHI     11325 DDX42 356 FLHDHQAEL    259197 NCR3 357 FLIQEIKTL     23352 UBR4 358 FLTDYLNDL     54880 BOOR 359 FMQDPMEVFV     10212 DDX39A 360 HLIDTNKIQL     55619 DOCK10 361 ILQEFESKL      2074, ERCC6, PGBD3    267004 362 ILTELGGFEV     51366 UBR5 363 ITTEVVNELYV     57222 ERGIC1 364 KMDWIFHTI      5290 PIK3CA 365 LISPLLLPV    144132 DNHD1 366 LLSETCVTI     23469 PHF3 367 NLWSLVAKV      9889 ZBED4 368 QLQPTDALLCV    125950 RAVER1 369 RLLDLENSLLGL     54899 PXK 370 SIFASPESV     10285 SMNDC1 371 SLADDSVLERL     23195 MDN1 372 SLFGPLPGPGPALV      9595 CYTIP 373 TLLADQGEIRV      9219 MTA2 374 VLSVITEEL     55621 TRMT1 375 VLWFKPVEL     55720 TSR1 376 VLYNQRVEEI      6730 5RP68 377 VVDGTCVAV     57589 KIAA1432 378 YILGKFFAL      8914 TIMELESS 379 YLAELVTPIL      1981 EIF4G1 380 YLDRKLLTL      6850 SYK 381 YLLEENKIKL    139818 DOCK11 382 YLLPLLQRL     55794 DDX28 383 YLLREWVNL     23019 CNOT1 384 YMIGSEVGNYL      6598 SMARCB1 385 YTIPLAIKL    128869 PIGU

TABLE 3  Peptides useful for e.g. personalized cancer therapies. SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s) 386 FLGDYVENL 54832 VPS13C 387 YLILSSHQL 1269 CNR2 388 GLLSALENV 1269 CNR2 389 GLAALAVHL 2175 FANCA 390 GLEERLYTA 29933 GPR132 391 FLLEREQLL 165055 CCDC138 392 FLWERPTLLV 79922 MRM1 393 FVMEGEPPKL 348654 GEN1 394 ILSTEIFGV 79703 C11orf80 395 ALYGKLLKL 157680 VPS13B 396 TLLGKQVTL 157680 VPS13B 397 FLPPEHTIVYI 9896 FIG4 398 FLAELPGSLSL 5326 PLAGL2 399 ALAAPDIVPAL 79886 CAAP1 400 ALFQPHLINV 10097 ACTR2 401 AMADKMDMSL 10189 ALYREF 402 LLVSNLDFGV 10189 ALYREF 403 FIMPATVADATAV 23352 UBR4 404 FLQPDLDSL 10514 MYBBP1A 405 FLVEKQPPQV 6778 STAT6 406 FLWPKEVEL 146206 RLTPR 407 FMIDASVHPTL 221960, 51622 CCZ1B, CCZ1 408 FMVDNEAIYDI 10376, 112714, TUBA1B, TUBA3E, TUBA3D,  113457, 51807, 7277, TUBA8, TUBA4A, TUBA3C,  7278, 7846, 84790 TUBA1A, TUBA1C 409 GLDAATATV 9869 SETDB1 410 GLFDGVPTTA 122618 PLD4 411 GLFEDVTQPGILL 23140 ZZEF1 412 ILGTEDLIVEV 79719 AAGAB 413 ILLEHGADPNL 22852 ANKRD26 414 ILSVVNSQL 80183 KIAA0226L 415 ILVTSIFFL 643 CXCR5 416 IMEDIILTL 1656 DDX6 417 IQIGEETVITV 2316 FLNA 418 IVTEIISEI 64151 NCAPG 419 LLNEILEQV 64151 NCAPG 420 KLIDDQDISISL 80208 SPG11 421 KLWTGGLDNTV 7088, 7090, 7091 TLE1, TLE3, TLE4 422 KQFEGTVEI 675 BRCA2 423 LLIGTDVSL 9730 VPRBP 424 LLPPLESLATV 5518 PPP2R1A 425 LLSDVRFVL 53339 BTBD1 426 LLWGNLPEI 653820, 729533 FAM72B, FAM72A 427 LLYDAVHIV 2899 GRIK3 428 LMYPYIYHV 54954 FAM120C 429 NLLETKLQL 219988 PATL1 430 QLIEKNWLL 56992 KIF15 431 RMVAEIQNV 11262 5P140 432 SAVDFIRTL 9263 STK17A 433 SILDRDDIFV 8237 USP11 434 SIQQSIERLLV 4809 NHP2L1 435 SLFNQEVQI 100528032, 22914, KLRK1, KLRC4 8302 436 SLFSSLEPQIQPV 23029 RBM34 437 SLEPQIQPV 23029 RBM34 438 SLFIGEKAVLL 23029 RBM34 439 SLLDLHTKV 27340 UTP20 440 SLLEQGKEPWMV 147949, 163087, ZNF583, ZNF383, ZNF850,  342892, 374899, ZNF829, ZNF527 84503 441 SLLEVNEASSV 149628 PYHIN1 442 SLNDLEKDVMLL 6597 SMARCA4 443 SLTIDGIYYV 1659 DHX8 444 SMSGYDQVL 3187, 3188 HNRNPH1, HNRNPH2 445 VIIKGLEEITV 3832 KIF11 446 VILTSSPFL 10800 CYSLTR1 447 VLDDSIYLV 57565 KLHL14 448 VMNDRLYAI 57565 KLHL14 449 LLDAMNYHL 57565 KLHL14 450 VLGPGPPPL 254359 ZDHHC24 451 VLIEYNFSI 55215 FANCI 452 VLLSLLEKV 1130 LYST 453 SLLPLVWKI 1130 LYST 454 VLMDEGAVLTL 54596 L1TD1 455 VLPDPEVLEAV 57326 PBXIP1 456 YAAPGGLIGV 1968, 255308 EIF2S3 457 YIMEPSIFNTL 51497 TH1L 458 YIQEHLLQI 10625 IVNS1ABP 459 YLDFSNNRL 51284 TLR7 460 YLEDGFAYV 5558 PRIM2 461 YLLNLNHLGL 23471 TRAM1 462 YVLPKLYVKL 100128168,  RPS26P39, RPS26P11, 100996747, 441502, RPS26, RPS26P28, 6231, 643003, RPS26P20, RPS26P15, 644166, 644928, RPS26P50, RPS26P2,  644934, 646753, RPS26P25, RPS26P58 728937, 729188 463 ALMAVVSGL 55103 RALGPS2 464 ALSDETKNNWEV 5591 PRKDC 465 FLYDEIEAEVNL 23191, 26999 CYFIP1, CYFIP2 466 FLYDEIEAEV 23191, 26999 CYFIP1, CYFIP2 467 IMQDFPAEIFL 25914 RTTN 468 KIQEILTQV 10643 IGF2BP3 469 SLDGTELQL 284114 TMEM102 470 TLTNIIHNL 94101 ORMDL1 471 VLLAAGPSAA 23225 NUP210 472 YLFESEGLVL 283431 GAS2L3 473 ALADDDFLTV 4173 MCM4 474 ALADLIEKELSV 4850 CNOT4 475 ALDDMISTL 7203 CCT3 476 ALITEVVRL 26005 C2CD3 477 ALLDQTKTLAESAL 7094 TLN1 478 AMFESSQNVLL 64328 XPO4 479 YLAHFIEGL 64328 XPO4 480 FLAEDPKVTL 60489 APOBEC3G 481 FLIDTSASM 203522, 26512 DDX26B, INTS6 482 FLTDLEDLTL 26151 NAT9 483 FLTEMVHFI 93517 SDR42E1 484 FLVDGPRVQL 90204 ZSWIM1 485 GLLDCPIFL 2177 FANCD2 486 GLLDLPFRVGV 23347 SMCHD1 487 GLSDGNPSL 79684 MSANTD2 488 GVYDGEEHSV 4113 MAGEB2 489 HLANIVERL 117854, 445372, TRIM6, TRIM6-TRIM34, 53840 TRIM34 490 ILDEKPVII 23460 ABCA6 491 ILFSEDSTKLFV 84916 CIRH1A 492 ILLDDNMQIRL 5261 PHKG2 493 LIIDQADIYL 27042 DIEXF 494 LLGDSSFFL 283254 HARBI1 495 LLLDEEGTFSL 27013 CNPPD1 496 LLVEQPPLAGV 4773 NFATC2 497 LLWDVPAPSL 1388, 7148 ATF6B, TNXB 498 LTAPPEALLMV 79050 NOC4L 499 NLMEMVAQL 55636 CHD7 500 QIITSVVSV 5378 PMS1 501 QLALKVEGV 25896 INTS7 502 QLLDETSAITL 10915 TCERG1 503 QLYEEPDTKL 10270 AKAP8 504 RLYSGISGLEL 84172 POLR1B 505 RVLEVGALQAV 25885 POLR1A 506 SLLPLDDIVRV 3708, 3709 ITPR1, ITPR2 507 SLLTEQDLWTV 90806 ANGEL2 508 SVDSAPAAV 10635 RAD51AP1 509 TLQEVVTGV 55750 AGK 510 TVDVATPSV 8924 HERC2 511 VLAYFLPEA 4171 MCM2 512 VLISVLQAI 26999 CYFIP2 513 YLIPFTGIVGL 26001 RNF167 514 YLLDDGTLVV 8872 CDC123 515 YLMPGFIHL 168400, 55510 DDX53, DDX43 516 YLPDIIKDQKA 5496 PPM1G 517 YLQLTQSEL 283237 TTC9C 518 YLTEVFLHVV 55024 BANK1 519 YLVEGRFSV 55125 CEP192 520 YLVYILNEL 51202 DDX47 521 YLYGQTTTYL 7153 TOP2A 522 YVLTQPPSV 28796, 28815, IGLV3-21, IGLV2-14,  28831, 3537, 3538 IGLJ3, IGLC1, IGLC2

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1V describe relative presentation of various peptides of the present disclosure.

FIGS. 2A-2D describe relative expression of various peptides of the present disclosure.

FIGS. 3A-3F describe simulation graphs of various peptides of the present disclosure.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention furthermore generally relates to the peptides according to the present invention for use in the treatment of proliferative diseases, such as, for example, acute myelogenous leukemia, bile duct cancer, brain cancer, breast cancer, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, urinary bladder cancer and uterine cancer.

Particularly preferred are the peptides—alone or in combination—according to the present invention selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385. More preferred are the peptides—alone or in combination—selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 202 (see Table 1), and their uses in the immunotherapy of CLL, acute myelogenous leukemia, bile duct cancer, brain cancer, breast cancer, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, urinary bladder cancer and uterine cancer, and preferably CLL.

As shown in the following Tables 4A and B, many of the peptides according to the present invention are also found on other tumor types and can, thus, also be used in the immunotherapy of other indications. Also, refer to FIG. 1 and Example 1.

TABLE 4A  Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases. The table shows for selected peptides on which additional tumor types they were found and either over-presented on more than 5% of the measured    tumor samples, or presented on more than 5% of the measured tumor samples  with a ratio of geometric means tumor vs normal tissues being  larger than 3.Over-presentation is defined as higher presentation on the  tumor sample as compared to the normal sample with highest presentation Normal tissues against which over-presentation was tested were: adipose tissue, adrenal gland, blood cells, blood vessel, bone marrow, brain, breast, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary gland, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter, and urinary bladder. SEQ ID No. Sequence Other relevant organs/diseases 1 AIPPSFASIFL NHL 2 ALHRPDVYL NHL 3 VIAELPPKV NHL 5 ALIFKIASA AML, BRCA 7 ALLERTGYTL HCC, NHL 8 ALAASALPALV AML 9 ALCDTLITV NHL 10 FVYGESVEL NHL 12 ALGEDEITL NHL, AML, Melanoma 13 VVDGMPPGV NHL 14 ALLRLLPGL NHL, AML 15 ALPEVSVEA NHL, AML, Melanoma 19 QVMEKLAAV NHL 20 ALVDPGPDFVV NHL 21 ALWAGLLTL NHL 22 ALWDPVIEL NHL 24 AMAGDVVYA NHL 25 ATYSGLESQSV Melanoma 26 AVLLVLPLV NHL 27 AVLGLVWLL NSCLC, NHL, Uterine Cancer 28 AVLHLLLSV NHL, BRCA 29 AVLQAVTAV SCLC, NHL, AML, Melanoma, OC, Gallbladder Cancer,  Bile Duct Cancer 30 ELLEGSEIYL NHL 32 FIDKFTPPV NHL 33 FIINSSNIFL NHL 35 FILPSSLYL RCC, NHL, AML, Gallbladder Cancer, Bile Duct Cancer 37 FIMEGGAMVL AML 38 FIMEGGAMV NHL 39 FLDALLTDV SCLC, NHL, AML, Melanoma 40 FLDEDDMSL SCLC, NHL, Urinary bladder cancer 42 FLEEGGVVTV NHL, Melanoma 43 FLLGPEALSFA NHL, AML 44 FLLSINDFL Melanoma 45 FLPELPADLEA NHL, Melanoma 46 YIIDSAQAV NHL 48 FLSPQQPPLL NHL 49 FLTDLFAQL NHL 52 FLVETGFLHV Melanoma 55 FMEPTLLML NHL, Urinary bladder cancer 56 FVFEAPYTL NHL, AML 57 GLSEISLRL NHL, AML, Melanoma 58 YIQQGIFSV NHL 59 FVFGDENGTVSL NHL, Esophageal Cancer, Urinary bladder cancer,  Uterine Cancer 60 FVLDHEDGLNL Melanoma, Esophageal Cancer 62 GIIDGSPRL NHL 63 SLAHVAGCEL NHL 64 KLLESVASA NHL 66 SLAGGLDDMKA NHL 67 GLDDVTVEV HCC, NHL, Melanoma, Esophageal Cancer, OC,  Gallbladder Cancer, Bile Duct Cancer 68 GLDQQFAGLDL RCC, HCC, NHL, Melanoma 69 GLHQREIFL NHL 70 FVPDTPVGV NHL 71 GLKHDIARV NHL 72 GLLDAGKMYV NHL, Melanoma 74 GLLRASFLL NHL, AML, BRCA 75 GLSIFAQDLRL NHL 76 GLLRIIPYL NHL 78 GLPSFLTEV HCC, NHL, AML, Melanoma 79 GLQAKIQEA NHL, Esophageal Cancer, Uterine Cancer 80 VLIEDELEEL NHL 81 WLVGQEFEL NHL 82 GLQSGVDIGV NHL 83 GQGEVLVYV SCLC, GC, Esophageal Cancer 86 HLYPGAVTI HCC, NHL 87 HQIEAVDGEEL GC, NHL, Esophageal Cancer 88 ILDEIGADVQA PC, NHL 89 ILDFGTFQL NHL 91 ILDLNTYNV NHL 92 ILEPLNPLL NHL 93 ILFNTQINI SCLC, NHL, AML, Melanoma, OC 94 ILFPLRFTL NSCLC, RCC, CRC, NHL, Melanoma, OC 96 ILIDKTSFV NHL 97 LLFATQITL NHL 98 SLIKYFLFV NHL 99 ILIFHSVAL NHL, AML 100 ILNNEVFAI NHL, AML 101 ILVVIEPLL NHL 102 IQDRAVPSL AML 103 KLGGTPAPA NHL 105 KLMNDIADI NHL 109 KLWEGLTELV AML 110 LLFDHLEPMEL NHL 111 KLWNVAAPLYL OC 112 KTLDVDATYEI HCC, NHL, Melanoma, Esophageal Cancer, OC 113 KVPAEEVLVAV NHL 116 LLIGATMQV SCLC, NHL, AML 117 LLILENILL NHL, AML, Melanoma 118 RLLILENILL NHL 119 VLPAEFFEV NHL, AML, Urinary bladder cancer, Uterine Cancer 120 AIDAALTSV NHL, AML 121 RLYEITIEV SCLC, NHL, AML, BRCA, OC 122 LLIPVVPGV NSCLC, NHL 124 LLLEETEKQAV RCC, HCC 125 LLLEIGEVGKLFV NHL 126 LLPEGGITAI NHL 127 LLPTAPTTV NHL 130 LLVLIPVYL NHL, Melanoma 133 YLLSGDISEA NHL 136 NLDKLVVTLV GC, Esophageal Cancer 137 NLIKTVIKL NHL 138 NLLDIDAPVTV SCLC, NHL 139 NLTDVVEKL RCC, NHL 141 QILSEIVEA NHL, Melanoma 145 SALDTITTV NHL 146 SIIEGPIIKL NHL, Urinary bladder cancer 147 SILETVATL NHL, AML 148 SIVASLITV NHL, AML 149 SLDNGGYYI NHL 150 SLFDQPLSII NHL 151 SLFDSAYGA AML 152 SLIRILQTI HCC 153 SLLAELHVL NHL 154 SLLAELHVLTV NHL 155 SLMLEVPAL HCC, NHL 156 SLNIGDVQL NHL 157 SLNIRDFTM AML 158 SLPEAPLDV NHL, AML, Uterine Cancer 159 SLQEEKLIYV Brain Cancer, NHL, MCC, Melanoma, Esophageal Cancer, Urinary bladder cancer 160 SLSFLVPSL NHL, AML, Urinary bladder cancer 161 SMDDGMINV CRC, NHL, Gallbladder Cancer, Bile Duct Cancer 162 SMKDDLENV NSCLC, SCLC, RCC, HCC, NHL, BRCA, Melanoma, Esophageal Cancer, Uterine Cancer, Gallbladder  Cancer, Bile Duct Cancer 163 SQLDISEPYKV NHL 164 SVHKGFAFV NSCLC, RCC, Brain Cancer, GC, AML, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer 165 TLDDDLDTV NHL 166 TLDPNQVSL AML 167 TLDTSKLYV NHL 168 YLLDQSFVM NHL 169 TLLLGLTEV NHL, Melanoma 170 TLTFRVETV NHL, Melanoma 172 TLYDMLASI CRC, NHL, AML, Melanoma 175 FTVPRVVAV NHL 177 VLDDRELLL NHL 178 VLFFNVQEV SCLC, NHL 179 VLLGLEMTL NHL, Esophageal Cancer 180 LLKDGPEIGL NHL 181 VLLSIPFVSV HCC, NHL 183 VLMPTVYQQGV AML 185 VMDDQRDLI AML 186 VMDPTKILI AML, Urinary bladder cancer 187 VMDTHLVNI NHL, Urinary bladder cancer 188 VMGDIPAAV NHL, AML, Gallbladder Cancer, Bile Duct Cancer 189 VMLEMTPEL SCLC, PC, NHL, BRCA, Melanoma, Gallbladder Cancer,  Bile Duct Cancer 190 VVMGTVPRL NHL, AML 192 YIQEYLTLL NHL 193 YLDLSNNRL NHL 194 YLDNVLAEL NHL 195 YLGGFALSV NHL, AML 196 YLLLQTYVL NHL 197 YLQEVPILTL NHL, Melanoma 199 YLVELSSLL HCC, NHL, AML 200 YMFEEVPIVI NSCLC, SCLC, CRC, HCC, NHL, Melanoma, Esophageal Cancer, OC, Urinary bladder cancer, Gallbladder   Cancer, Bile Duct Cancer 201 YQLELHGIEL Esophageal Cancer 202 YVDDVFLRV Urinary bladder cancer 203 ALLSSQLAL NHL 204 GLLQINDKIAL SCLC, RCC, HCC, Melanoma, OC, Urinary bladder cancer 205 GLSQANFTL NHL 207 IIADLDTTIMFA SCLC, NHL, Melanoma, OC 208 ILLKTEGINL NHL, BRCA, OC, Urinary bladder cancer 209 ILQAELPSL NHL 210 KLLVQDFFL HCC, NHL 211 LIDVKPLGV NHL, BRCA, Melanoma 212 NIIEAINELLV NHL, BRCA, Melanoma 213 RLLYQLVFL NHL, Esophageal Cancer, Urinary bladder cancer,  Gallbladder Cancer, Bile Duct Cancer 214 RLQELTEKL NHL, AML 215 VMQDIVYKL NHL, Esophageal Cancer, Urinary bladder cancer 216 WLAGDVPAA SCLC, NHL 217 ALDEPPYLTV NHL, Melanoma 218 ALGEEWKGYVV Esophageal Cancer 219 ALLNLLESA NHL, AML, OC 220 ALPEILFAKV NHL 221 ALVSTIIMV NHL, AML 222 ALWELSLKI NHL 223 ALWVSQPPEI NHL 224 AMEALVVEV NHL 225 ILQERELLPV NHL, Melanoma, OC, Urinary bladder cancer 227 FLAEASVMTQL NHL 228 FLGGLSPGV NHL, Esophageal Cancer 229 FLLNLQNCHL NHL 230 FLQDSKVIFV NHL 231 FLYIRQLAI AML 232 FMHQIIDQV RCC, CRC, Melanoma 233 GIIDINVRL PC, NHL, Melanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 234 GLDDAEYAL NHL, AML, Melanoma, Urinary bladder cancer 235 GLDDLLLFL SCLC, PC, NHL, AML, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 236 GLLESGRHYL NHL, AML 237 GLQENLDVVV SCLC, NHL, Melanoma, Uterine Cancer 238 GLVETELQL NHL, AML 239 ILAGEMLSV NHL, AML, Melanoma, Gallbladder Cancer, Bile Duct Cancer 240 ILARDILEI NHL, Urinary bladder cancer 241 ILGDILLKV AML 242 ILLGIQELL NHL, AML 243 ILPTLEKELFL AML 244 ILQALAVHL NHL, AML, OC 245 KIMDYSLLLGV SCLC, RCC, OC 246 KLDETGVAL HCC, NHL, AML, Melanoma 247 KLKDRLPSI NHL 248 KTVEPPISQV NHL 249 LLPTGVFQV Urinary bladder cancer 250 LLVQEPDGLMV SCLC, HCC, Melanoma, Urinary bladder cancer 251 LLYDNVPGA NHL 252 NLLDPGSSYLL Esophageal Cancer 253 NLWSVDGEVTV NHL 254 QLIPKLIFL NHL, Melanoma 255 YLFEEAISM NHL 257 RIINGIIISV NHL 258 RLDYITAEI RCC, NHL, AML, Melanoma 260 SLDDVEGMSV NHL, Gallbladder Cancer, Bile Duct Cancer 261 SLVEAQGWLV NHL 263 SQWEDIHVV NHL, AML 264 TILDYINVV Brain Cancer, NHL, AML, BRCA, OC, Uterine Cancer 265 TLLADDLEIKL Esophageal Cancer 266 TLLDQLDTQL NHL, OC, Urinary bladder cancer 267 TLLDWQDSL NHL, AML, Melanoma 268 TLLQVFHLL NHL 269 TLTDEQFLV NHL, AML 271 VIRNIVEAA GC, CRC, PC, AML, Esophageal Cancer 272 VLDELPPLI SCLC, NHL 273 VLGEYSYLL NHL 274 VLLEYHIAYL NSCLC, SCLC, HCC, NHL, Urinary bladder cancer 275 VLLFIEHSV NHL 276 VLNDGAPNV NSCLC, SCLC, NHL, Melanoma, Uterine Cancer,  Gallbladder Cancer, Bile Duct Cancer 278 YLDDLLPKL PC, NHL 280 YTLDSLYWSV NHL 281 NLLDDRGMTAL NHL, AML 282 LLRDGIELV NHL, Melanoma, OC 283 ILQPMDIHV NHL 284 LLSAAEPVPA NHL 286 FLLEDLSQKL NHL 288 GLAESTGLLAV NHL 290 LANPHELSL AML 291 ILLNEDDLVTI SCLC, NHL, Melanoma 292 AAALIIHHV Urinary bladder cancer 294 ALLDQLHTLL NHL 295 ALLQKLQQL NHL 296 FIAPTGHSL SCLC, NHL, AML, BRCA 297 FLVEPQEDTRL NHL, Melanoma 298 IILPVEVEV SCLC, NHL, AML, Urinary bladder cancer 299 ILEENIPVL NHL 300 ILLNPAYDVYL NHL, Melanoma 303 ILSQPTPSL NHL, AML, Melanoma, OC 304 LAIVPVNTL AML 305 LLFPQIEGIKI NHL, Melanoma 306 VVAEELENV NHL, Melanoma, OC 307 LLLTKPTEA NHL 308 SLYDVSRMYV NHL 309 ILYGTQFVL NHL 310 LLSTLHLLV NHL 311 LLVDVEPKV NSCLC, SCLC, RCC, HCC, PC, NHL, AML, Melanoma 312 LLYNSTDPTL NHL 313 LMADLEGLHL Melanoma, Urinary bladder cancer 314 LMKDCEAEV NHL 316 MLLEHGITL NHL 317 NLLAHIWAL AML 320 TIAPVTVAV NHL 321 RLLEFELAQL SCLC, CRC, NHL, OC 322 SLASIHVPL NHL 323 SLDLFNCEV NHL 324 SLYSALQQA NHL 325 TLENGVPCV NHL 326 VLAFLVHEL Esophageal Cancer 327 VLIKWFPEV NHL 328 VLLPQETAEIHL NHL, BRCA, Melanoma, OC, Urinary bladder cancer 329 VLMDGSVKL NHL 330 VLMWEIYSL NHL 332 VMIQHVENL NHL 333 VTLEFPQLIRV SCLC, HCC, NHL 334 YLLEEKIASL NHL 335 YLYQEQYFI SCLC, NHL, AML, Urinary bladder cancer 337 YMYEKESEL NHL 338 FLDMTNWNL HCC, NHL, Melanoma, OC, Urinary bladder cancer 339 GLWGTVVN INHL, AML, Melanoma, Uterine Cancer 341 LLAELPASVHA HCC, NHL, Urinary bladder cancer 342 SLITPLQAV NHL, AML, Urinary bladder cancer, Gallbladder  Cancer, Bile Duct Cancer 343 TLLEALDCI NHL, AML, Melanoma, Esophageal Cancer 344 VLAFENPQV Esophageal Cancer 346 VLVQVSPSL RCC, NHL, AML, Gallbladder Cancer, Bile Duct Cancer 347 YLGPVSPSL NHL 348 ALAKPPVVSV SCLC, HCC, Melanoma, Esophageal Cancer 349 ALATHILSL NHL, Urinary bladder cancer 350 ALEDRVWEL NHL, Esophageal Cancer 351 ALSEKLARL HCC, NHL, AML, Urinary bladder cancer 352 ALVFELHYV NHL 353 ATPMPTPSV SCLC, NHL 354 FIMDDPAGNSYL CRC, NHL, AML, Melanoma, Esophageal Cancer, Urinary 357 FLIQEIKTL bladder cancer, Gallbladder Cancer, Bile Duct Cancer NHL, OC, Urinary bladder cancer 358 FLTDYLNDL NHL 359 FMQDPMEVFV Melanoma 360 HLIDTNKIQL NHL 362 ILTELGGFEV NHL 363 ITTEVVNELYV NHL, Melanoma 364 KMDWIFHTI Urinary bladder cancer 365 LISPLLLPV NHL, AML, Melanoma, Urinary bladder cancer 367 NLWSLVAKV NSCLC, NHL, AML, Melanoma, OC 369 RLLDLENSLLGL AML 370 SIFASPESV Melanoma, Esophageal Cancer, Urinary bladder cancer 371 SLADDSVLERL RCC, HCC, NHL, AML, Esophageal Cancer, Urinary bladder cancer 373 TLLADQGEIRV NHL, Melanoma 374 VLSVITEEL NHL, AML 375 VLWFKPVEL NSCLC, HCC, NHL, Melanoma, Esophageal Cancer 376 VLYNQRVEEI Urinary bladder cancer 377 VVDGTCVAV NHL, Melanoma 378 YILGKFFAL NHL, AML 379 YLAELVTPIL NSCLC, SCLC, NHL, Melanoma 380 YLDRKLLTL NHL 381 YLLEENKIKL NHL 382 YLLPLLQRL NHL, AML 383 YLLREVVVNL HCC, NHL 384 YMIGSEVGNYL NHL, Urinary bladder cancer 385 YTIPLAIKL NHL, AML, BRCA, Esophageal Cancer NSCLC = non-small cell lung cancer, SCLC = small cell lung cancer, RC = kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer, HCC = liver cancer, PC = pancreatic cancer, PrC = prostate cancer, leukemia, BRCA = breast cancer, MCC = Merkel cell carcinoma, NHL = Non Hodgkin lymphoma

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 1, 2, 3, 7, 9, 10, 13, 14, 15, 19, 20, 21, 22, 24, 26, 27, 28, 29, 30, 32, 33, 35, 38, 39, 40, 42, 43, 45, 46, 48, 49, 55, 56, 57, 58, 59, 62, 63, 64, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 79, 80, 81, 82, 86, 87, 88, 89, 91, 92, 93, 94, 96, 97, 98, 99, 100, 101, 103, 105, 110, 112, 113, 116, 117, 118, 119, 120, 121, 122, 125, 126, 127, 130, 133, 137, 138, 139, 141, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 165, 167, 168, 169, 170, 172, 175, 177, 178, 179, 180, 181, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 199, 200, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 219, 220, 221, 222, 223, 224, 225, 227, 228, 229, 230, 233, 234, 235, 236, 237, 238, 239, 240, 242, 244, 246, 247, 248, 251, 253, 254, 255, 257, 258, 260, 261, 263, 264, 266, 267, 268, 269, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 286, 288, 290, 291, 294, 295, 296, 297, 298, 299, 300, 303, 305, 306, 307, 308, 309, 310, 311, 312, 314, 316, 320, 321, 322, 323, 324, 325, 327, 328, 329, 330, 332, 333, 334, 335, 337, 338, 339, 341, 342, 343, 346, 347, 349, 350, 351, 352, 353, 354, 357, 358, 360, 362, 363, 365, 367, 371, 373, 374, 375, 377, 378, 379, 380, 381, 382, 383, 384, and 385 for the—in one preferred embodiment combined—treatment of NHL.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 5, 8, 12, 14, 15, 29, 35, 37, 39, 43, 56, 57, 74, 78, 93, 99, 100, 102, 109, 116, 117, 119, 120, 121, 147, 148, 151, 157, 158, 160, 164, 166, 172, 183, 185, 186, 188, 190, 195, 199, 214, 219, 221, 231, 234, 235, 236, 238, 239, 241, 242, 243, 244, 246, 258, 263, 264, 267, 269, 271, 281, 290, 296, 298, 303, 304, 311, 317, 335, 339, 342, 343, 346, 351, 354, 365, 367, 369, 374, 378, 382, and 385 for the—in one preferred embodiment combined—treatment of AML.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 5, 28, 74, 121, 162, 208, 211, 212, 264, 296, 328, and 385 for the—in one preferred embodiment combined—treatment of BRCA.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 7, 67, 68, 78, 86, 112, 124, 152, 155, 162, 181, 199, 200, 204, 210, 246, 250, 274, 311, 333, 338, 341, 348, 351, 371, 375, and 383 for the—in one preferred embodiment combined—treatment of HCC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 12, 15, 25, 29, 39, 42, 44, 45, 52, 57, 60, 67, 68, 72, 78, 93, 94, 112, 117, 130, 141, 159, 162, 169, 170, 172, 189, 197, 200, 204, 207, 211, 212, 217, 225, 232, 233, 234, 237, 239, 246, 250, 254, 258, 267, 276, 282, 291, 297, 300, 303, 305, 306, 311, 313, 328, 338, 339, 343, 348, 354, 359, 363, 365, 367, 370, 373, 375, 377, and 379 for the—in one preferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 27, 94, 122, 162, 164, 200, 274, 276, 311, 367, 375, and 379 for the—in one preferred embodiment combined—treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 27, 59, 79, 119, 158, 162, 164, 233, 235, 237, 264, 276, and 339 for the—in one preferred embodiment combined—treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 29, 67, 93, 94, 111, 112, 200, 204, 207, 208, 219, 225, 244, 245, 264, 266, 282, 303, 306, 321, 328, 338, 357, and 367 for the—in one preferred embodiment combined—treatment of ovarian cancer (OC).

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 29, 35, 67, 161, 162, 188, 189, 200, 213, 233, 235, 239, 260, 276, 342, 346, and 354 for the—in one preferred embodiment combined—treatment of gallbladder cancer and/or bile duct cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 35, 68, 94, 124, 139, 162, 164, 197, 204, 232, 245, 258, 311, 346, and 371 for the—in one preferred embodiment combined—treatment of RCC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 39, 40, 83, 93, 121, 138, 162, 178, 189, 200, 204, 207, 216, 235, 237, 245, 250, 272, 274, 276, 291, 296, 298, 311, 321, 333, 335, 348, 353, and 379 for the—in one preferred embodiment combined—treatment of SCLC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 40, 55, 59, 119, 146, 159, 160, 164, 186, 187, 200, 202, 204, 208, 213, 215, 225, 233, 234, 235, 240, 249, 250, 266, 274, 292, 298, 313, 328, 335, 338, 341, 342, 349, 351, 354, 357, 364, 365, 370, 371, 376, and 384 for the—in one preferred embodiment combined—treatment of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 59, 60, 67, 79, 83, 87, 112, 136, 159, 162, 164, 179, 200, 201, 213, 215, 218, 228, 233, 252, 265, 271, 326, 343, 344, 348, 350, 354, 370, 371, 375, and 385 for the—in one preferred embodiment combined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 83, 87, 136, 164, and 271 for the—in one preferred embodiment combined—treatment of gastric cancer (GC).

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 88, 189, 233, 235, 271, 278, and 311 for the—in one preferred embodiment combined—treatment of pancreatic cancer (PC).

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 94, 161, 172, 200, 232, 271, 321, and 354 for the—in one preferred embodiment combined—treatment of colorectal cancer (CRC).

Thus, another aspect of the present invention relates to the use of the peptide according to the present invention according to SEQ ID No. 159 for the treatment of MCC.

Thus, another aspect of the present invention relates to the use of the peptide according to the present invention according to SEQ ID NO: 62 for the treatment of glioblastoma, renal cell carcinoma, hepatocellular carcinoma, and testis cancer.

Thus, another aspect of the present invention relates to the use of the peptides according to the present invention for the—preferably combined—treatment of a proliferative disease selected from the group of CLL, acute myelogenous leukemia, bile duct cancer, brain cancer, breast cancer, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, urinary bladder cancer and uterine cancer.

TABLE 4B  Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases.  The table shows, like Table 4A, for selected peptides on which additional      tumour types they were found showing over presentation (including specific  presentation) on more than 5% of the measured tumour samples, or   presentation on more than 5% of the measured tumour samples with a ratio of  geometric means tumour vs normal tissues being larger than 3. Over- presentation is defined as higher presentation on the tumour sample as  as compared to the normal sample with highest presentation. Normal tissues against which over-presentation was tested were: adipose  tissue, adrenal gland, artery, bone marrow, brain, central nerve, colon,  duodenum, oesophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear white blood cells, pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary, pleura, rectum, salivary gland, skeletal  muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea,  ureter, urinary bladder, vein.  SEQ ID NO. Sequence Additional Entities 5 ALIFKIASA Uterine Cancer 7 ALLERTGYTL Uterine Cancer, AML 8 ALAASALPALV AML, HNSCC 11 ALFTFSPLTV AML, HNSCC, NHL, NSCLC, OC, SCLC 13 VVDGMPPGV BRCA, Uterine Cancer 14 ALLRLLPGL BRCA, Melanoma, HNSCC 15 ALPEVSVEA Esophageal Cancer 25 ATYSGLESQSV AML 26 AVLLVLPLV NHL, NSCLC 27 AVLGLVWLL HNSCC 29 AVLQAVTAV Uterine Cancer 30 ELLEGSEIYL CRC, BRCA, Melanoma, Uterine Cancer 35 FILPSSLYL SCLC, Uterine Cancer 37 FIMEGGAMVL NHL 38 FIMEGGAMV HNSCC 40 FLDEDDMSL HNSCC 41 FLDPSLDPLL Uterine Cancer 42 FLEEGGVVTV SCLC, AML 44 FLLSINDFL RCC, Uterine Cancer, AML, NHL 45 FLPELPADLEA NSCLC, Uterine Cancer, OC 48 FLSPQQPPLL HNSCC 49 FLTDLFAQL NHL, Melanoma 51 FLVEAPHDWDL Uterine Cancer 55 FMEPTLLML HNSCC 58 YIQQGIFSV NHL, NSCLC 59 FVFGDENGTVSL SCLC, HNSCC 67 GLDDVTVEV AML 68 GLDQQFAGLDL BRCA, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, HNSCC 76 GLLRIIPYL RCC 82 GLQSGVDIGV AML, Esophageal Cancer, PC 83 GQGEVLVYV NHL 84 GVMDVNTAL AML, HCC, HNSCC, NHL, NSCLC, GC, RCC 86 HLYPGAVTI AML 87 HQIEAVDGEEL PC 89 ILDFGTFQL BRCA 90 VIADLGLIRV Melanoma 91 ILDLNTYNV AML 93 ILFNTQINI Uterine Cancer, HNSCC 94 ILFPLRFTL Gallbladder Cancer, Bile Duct Cancer, AML, HNSCC 101 ILVVIEPLL Uterine Cancer 107 ILYNLYDLL Melanoma 108 VIYTLIHYI BRCA, Melanoma 111 KLWNVAAPLYL Melanoma 112 KTLDVDATYEI Uterine Cancer 113 KVPAEEVLVAV NHL, NSCLC 114 LIPEGPPQV AML, HNSCC 115 LLFDKLYLL Uterine Cancer 118 RLLILENILL HNSCC 119 VLPAEFFEV SCLC 124 LLLEETEKQAV HNSCC 125 LLLEIGEVGKLFV BRCA 126 LLPEGGITAI SCLC, HCC, Melanoma, Uterine Cancer, HNSCC 127 LLPTAPTTV SCLC 128 LLSEEEYHL Urinary Bladder Cancer, NHL, HNSCC 130 LLVLIPVYL Uterine Cancer 136 NLDKLWTLV AML, CRC, Melanoma, NSCLC, Brain Cancer 138 NLLDIDAPVTV Uterine Cancer, AML 141 QILSEIVEA Uterine Cancer 145 SALDTITTV NSCLC, RCC, GC, HCC, AML 146 SIIEGPIIKL HNSCC 147 SILETVATL Melanoma 152 SLIRILQTI SCLC 156 SLNIGDVQL AML 159 SLQEEKLIYV HNSCC 160 SLSFLVPSL Uterine Cancer 161 SMDDGMINV SCLC, Melanoma, Uterine Cancer, AML, HNSCC 162 SMKDDLENV PC, OC, HNSCC 164 SVHKGFAFV CRC, Melanoma, HNSCC 172 TLYDMLASI SCLC, Urinary Bladder Cancer, Uterine Cancer 173 TVIENIHTI CRC, HCC, Melanoma, NHL, NSCLC, OC,  Esophageal Cancer, GC, RCC 174 VLAELPIIVV NSCLC, OC 176 VLAEQNIIPSA NSCLC, SCLC, GC, Melanoma, NHL, HNSCC 179 VLLGLEMTL HNSCC 181 VLLSIPFVSV Melanoma, Gallbladder Cancer, Bile Duct Cancer 184 VLSHNLYTV NHL 187 VMDTHLVNI CRC, Melanoma, Uterine Cancer 188 VMGDIPAAV Urinary Bladder Cancer, HNSCC 189 VMLEMTPEL HNSCC 190 VVMGTVPRL Melanoma 191 YIFDGSDGGV Uterine Cancer 192 YIQEYLTLL SCLC, Urinary Bladder Cancer, AML, HNSCC 197 YLQEVPILTL Uterine Cancer 198 YLTFLPAEV BRCA, Uterine Cancer 199 YLVELSSLL Melanoma 200 YMFEEVPIVI BRCA, HNSCC 201 YQLELHGIEL NHL 202 YVDDVFLRV AML, HNSCC 203 ALLSSQLAL NHL, AML, NSCLC 204 GLLQINDKIAL BRCA, Uterine Cancer, AML 208 ILLKTEGINL Melanoma, HNSCC 211 LIDVKPLGV RCC, Brain Cancer, HCC, Esophageal Cancer,  HNSCC 212 NIIEAINELLV Uterine Cancer, AML, HNSCC 215 VMQDIVYKL HNSCC 216 WLAGDVPAA AML, HNSCC 217 ALDEPPYLTV AML 218 ALGEEWKGYVV AML 221 ALVSTIIMV Brain Cancer 224 AMEALVVEV AML 226 AMNISVPQV AML, PrC, Brain Cancer, Gallbladder Cancer, CCC,  HNSCC, NHL, NSCLC, OC, SCLC, Uterine Cancer 228 FLGGLSPGV SCLC, BRCA, Uterine Cancer, AML, HNSCC 231 FLYIRQLAI Uterine Cancer 232 FMHQIIDQV Uterine Cancer 234 GLDDAEYAL Uterine Cancer, HNSCC 235 GLDDLLLFL BRCA, HNSCC 237 GLQENLDVVV AML 239 ILAGEMLSV SCLC, HCC 240 ILARDILEI Melanoma 242 ILLGIQELL BRCA 247 KLKDRLPSI HCC 257 RIINGIIISV AML 258 RLDYITAEI GC, Uterine Cancer 260 SLDDVEGMSV SCLC, HNSCC 261 SLVEAQGWLV AML 264 TILDYINVV HNSCC 266 TLLDQLDTQL CRC, BRCA, Uterine Cancer 268 TLLQVFHLL Uterine Cancer 269 TLTDEQFLV BRCA 272 VLDELPPLI BRCA, Uterine Cancer, HNSCC 273 VLGEYSYLL NHL, HNSCC 274 VLLEYHIAYL Melanoma, HNSCC 275 VLLFIEHSV Melanoma 279 YMAPEVVEA HNSCC, NHL, NSCLC, SCLC, Uterine Cancer 280 YTLDSLYWSV SCLC, BRCA, Melanoma, HNSCC 286 FLLEDLSQKL Uterine Cancer 289 ILEEQPMDMLL Uterine Cancer 293 ALDIMIPMV AML, CRC, Brain Cancer, Gallbladder Cancer, CCC,  HNSCC, Melanoma, NHL, NSCLC, OC, Brain Cancer, RCC, SCLC, Urinary Bladder Cancer,  Uterine Cancer 294 ALLDQLHTLL AML 298 IILPVEVEV CRC, HNSCC 299 ILEENIPVL Uterine Cancer, AML 303 ILSQPTPSL Uterine Cancer 305 LLFPQIEGIKI Urinary Bladder Cancer, HNSCC 306 VVAEELENV RCC 311 LLVDVEPKV Uterine Cancer, HNSCC 312 LLYNSTDPTL Urinary Bladder Cancer 313 LMADLEGLHL Uterine Cancer, AML 317 NLLAHIWAL NHL 321 RLLEFELAQL Brain Cancer, PrC, Uterine Cancer, AML, HNSCC 323 SLDLFNCEV Uterine Cancer, AML 324 SLYSALQQA SCLC, Uterine Cancer, AML 326 VLAFLVHEL Esophageal Cancer, NSCLC, RCC 327 VLIKWFPEV Uterine Cancer 333 VTLEFPQLIRV BRCA 334 YLLEEKIASL AML 335 YLYQEQYFI CRC, Brain Cancer, Gallbladder Cancer, CCC,  NSCLC, UterineCancer 336 YMAVTTQEV Uterine Cancer 339 GLWGTVVNI HNSCC 340 KLLEEICNL Melanoma, Uterine Cancer, AML, NHL 342 SLITPLQAV HNSCC 343 TLLEALDCI GC, Uterine Cancer, HNSCC 344 VLAFENPQV HNSCC 346 VLVQVSPSL HNSCC 348 ALAKPPVVSV NSCLC, Urinary Bladder Cancer, NHL, HNSCC 349 ALATHILSL HCC, Melanoma 351 ALSEKLARL CRC, HNSCC, Melanoma, NSCLC, OC, PC, Brain Cancer, RCC, Uterine Cancer 353 ATPMPTPSV BRCA, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, HNSCC 354 FIMDDPAGNSYL Brain Cancer, HNSCC 355 FIWPMLIHI Melanoma, Uterine Cancer 357 FLIQEIKTL HCC 358 FLTDYLNDL AML 359 FMQDPMEVFV Melanoma, NHL, NSCLC, OC 360 HLIDTNKIQL NHL, NSCLC 361 ILQEFESKL AML, NHL 362 ILTELGGFEV AML, PrC, BRCA, PC, CRC, HCC, HNSCC,  Melanoma, NSCLC, OC, Esophageal Cancer, GC,  RCC, SCLC, Urinary Bladder Cancer, Uterine Cancer 363 ITTEVVNELYV BRCA 365 LISPLLLPV SCLC, Uterine Cancer, HNSCC 367 NLWSLVAKV Uterine Cancer 368 QLQPTDALLCV Uterine Cancer 370 SIFASPESV Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, HNSCC 374 VLSVITEEL SCLC, HCC, PrC, Urinary Bladder Cancer, Uterine Cancer 375 VLWFKPVEL BRCA, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, HNSCC 377 VVDGTCVAV BRCA, Uterine Cancer, HNSCC 378 YILGKFFAL Melanoma, Esophageal Cancer 379 YLAELVTPIL HCC, BRCA, Uterine Cancer, AML, HNSCC 383 YLLREWVNL Melanoma, Uterine Cancer 384 YMIGSEVGNYL SCLC, Uterine Cancer 385 YTIPLAIKL CRC NSCLC = non-small cell lung cancer, SCLC = small cell lung cancer, RCC = kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer, HCC = liver cancer, PC = pancreatic cancer, PrC = prostate cancer, BRCA = breast cancer, NHL = non-Hodgkin lymphoma, AML = acute myeloid leukemia, OC = ovarian cancer, HNSCC = head and neck squamous cell carcinoma, CCC = cholangiocarcinoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 5, 7, 13, 29, 30, 35, 41, 44, 45, 51, 68, 93, 101, 112, 115, 126, 130, 138, 141, 160, 161, 172, 187, 191, 197, 198, 204, 212, 226, 228, 231, 232, 234, 258, 266, 268, 272, 279, 286, 289, 293, 299, 303, 311, 313, 321, 323, 324, 327, 335, 336, 340, 343, 351, 353, 355, 362, 365, 367, 368, 370, 374, 375, 377, 379, 383, and 384 for the—in one preferred embodiment combined—treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 7, 8, 11, 25, 42, 44, 67, 82, 84, 86, 91, 94, 114, 136, 138, 145, 156, 161, 192, 202, 203, 204, 212, 216, 217, 218, 224, 226, 237, 257, 261, 293, 294, 299, 313, 321, 323, 324, 334, 340, 353, 358, 361, 362, 370, and 379 for the—in one preferred embodiment combined—treatment of AML.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 8, 11, 14, 27, 38, 40, 48, 55, 59, 68, 84, 93, 94, 114, 118, 124, 126, 128, 146, 159, 161, 162, 164, 176, 179, 188, 189, 192, 200, 202, 208, 211, 212, 215, 216, 226, 228, 234, 235, 260, 264, 272, 273, 274, 279, 280, 293, 298, 305, 311, 321, 339, 342, 343, 344, 346, 348, 351, 353, 354, 362, 365, 370, 375, 377, and 379 for the—in one preferred embodiment combined—treatment of HNSCC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 11, 26, 37, 44, 49, 58, 83, 84, 113, 128, 173, 176, 184, 201, 203, 226, 273, 279, 293, 317, 340, 348, 359, 360, and 361 for the—in one preferred embodiment combined—treatment of NHL.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 11, 26, 45, 58, 84, 113, 136, 145, 173, 174, 176, 203, 226, 279, 293, 326, 335, 348, 351, 359, 360, and 362 for the—in one preferred embodiment combined—treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 11, 45, 68, 162, 173, 174, 226, 293, 351, 359, 362, and 375 for the—in one preferred embodiment combined—treatment of OC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 11, 35, 42, 59, 119, 126, 127, 152, 161, 172, 176, 192, 226, 228, 239, 260, 280, 293, 324, 362, 365, 374, and 384 for the—in one preferred embodiment combined—treatment of SCLC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 13, 14, 30, 68, 89, 108, 125, 198, 200, 204, 228, 235, 242, 266, 269, 272, 280, 333, 353, 362, 363, 375, 377, and 379 for the—in one preferred embodiment combined—treatment of BRCA.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 14, 30, 49, 90, 107, 108, 111, 126, 136, 147, 161, 164, 173, 176, 181, 187, 190, 199, 208, 240, 274, 275, 280, 293, 340, 349, 351, 355, 359, 362, 378, and 383 for the—in one preferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 15, 82, 173, 211, 326, 362, and 378 for the—in one preferred embodiment combined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 30, 136, 164, 173, 187, 266, 293, 298, 335, 351, 362, 363, and 385 for the—in one preferred embodiment combined—treatment of CRC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 44, 76, 84, 145, 173, 211, 293, 306, 326, 351, and 362 for the—in one preferred embodiment combined—treatment of RCC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 68, 94, 181, 226, 293, 335, 353, 370, and 375 for the—in one preferred embodiment combined—treatment of gallbladder cancer and/or bile duct cancer and/or cholangiocarcinoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 82, 87, 162, 351, and 362 for the—in one preferred embodiment combined—treatment of PC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 84, 145, 173, 176, 258, 343, and 362 for the—in one preferred embodiment combined—treatment of GC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 84, 126, 145, 173, 211, 239, 247, 349, 357, 362, 374, and 379 for the—in one preferred embodiment combined—treatment of HCC.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 136, 211, 221, 226, 293, 321, 335, 351, and 354 for the—in one preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 128, 172, 188, 192, 293, 305, 312, 321, 348, 362, and 374 for the—in one preferred embodiment combined—treatment of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 226, 321, 362, and 374 for the—in one preferred embodiment combined—treatment of PrC.

The present invention furthermore relates to peptides according to the present invention that have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I or—in an elongated form, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to the present invention wherein said peptides (each) consist or consist essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 385.

The present invention further relates to the peptides according to the present invention, wherein said peptide is modified and/or includes non-peptide bonds.

The present invention further relates to the peptides according to the present invention, wherein said peptide is part of a fusion protein, in particular fused to the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or fused to (or into the sequence of) an antibody, such as, for example, an antibody that is specific for dendritic cells.

The present invention further relates to a nucleic acid, encoding the peptides according to the present invention. The present invention further relates to the nucleic acid according to the present invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable of expressing and/or expressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to the present invention, a nucleic acid according to the present invention or an expression vector according to the present invention for use in the treatment of diseases and in medicine, in particular in the treatment of cancer.

The present invention further relates to antibodies that are specific against the peptides according to the present invention or complexes of said peptides according to the present invention with MHC, and methods of making these.

The present invention further relates to T-cell receptors (TCRs), in particular soluble TCR (sTCRs) and cloned TCRs engineered into autologous or allogeneic T cells, and methods of making these, as well as NK cells or other cells bearing said TCR or cross-reacting with said TCRs.

The antibodies and TCRs are additional embodiments of the immunotherapeutic use of the peptides according to the invention at hand.

The present invention further relates to a host cell comprising a nucleic acid according to the present invention or an expression vector as described before. The present invention further relates to the host cell according to the present invention that is an antigen presenting cell, and preferably is a dendritic cell.

The present invention further relates to a method for producing a peptide according to the present invention, said method comprising culturing the host cell according to the present invention, and isolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to the present invention, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting a sufficient amount of the antigen with an antigen-presenting cell.

The present invention further relates to the method according to the present invention, wherein the antigen-presenting cell comprises an expression vector capable of expressing or expressing said peptide containing SEQ ID No. 1 to SEQ ID No.: 385, preferably containing SEQ ID No. 1 to SEQ ID No. 202, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced by the method according to the present invention, wherein said T cell selectively recognizes a cell which expresses a polypeptide comprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing target cells in a patient which target cells aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, the method comprising administering to the patient an effective number of T cells as produced according to the present invention.

The present invention further relates to the use of any peptide as described, the nucleic acid according to the present invention, the expression vector according to the present invention, the cell according to the present invention, the activated T lymphocyte, the T cell receptor or the antibody or other peptide- and/or peptide-MHC-binding molecules according to the present invention as a medicament or in the manufacture of a medicament. Preferably, said medicament is active against cancer.

Preferably, said medicament is a cellular therapy, a vaccine or a protein based on a soluble TCR or antibody.

The present invention further relates to a use according to the present invention, wherein said cancer cells are CLL, acute myelogenous leukemia, bile duct cancer, brain cancer, breast cancer, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, urinary bladder cancer and uterine cancer, and preferably CLL cells.

The present invention further relates to biomarkers based on the peptides according to the present invention, herein called “targets”, that can be used in the diagnosis of cancer, preferably CLL. The marker can be over-presentation of the peptide(s) themselves, or over-expression of the corresponding gene(s). The markers may also be used to predict the probability of success of a treatment, preferably an immunotherapy, and most preferred an immunotherapy targeting the same target that is identified by the biomarker. For example, an antibody or soluble TCR can be used to stain sections of the tumor to detect the presence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as an immune stimulating domain or toxin. The present invention also relates to the use of these novel targets in the context of cancer treatment.

Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor associated antigens has raised the possibility of using a host's immune system to intervene in tumor growth. Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of T-cells from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in natural immune defense against cancer. CD8-positive T-cells in particular, which recognize class I molecules of the major histocompatibility complex (MHC)-bearing peptides of usually 8 to 10 amino acid residues derived from proteins or defect ribosomal products (DRIPS) located in the cytosol, play an important role in this response. The MHC-molecules of the human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined as given below.

The term “T-cell response” means the specific proliferation and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I restricted cytotoxic T cells, effector functions may be lysis of peptide-pulsed, peptide-precursor pulsed or naturally peptide-presenting target cells, secretion of cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion of effector molecules, preferably granzymes or perforins induced by peptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids in length, and as long as 10, 11, 12, 13, or 14 or longer, and in case of MHC class II peptides (elongated variants of the peptides of the invention) they can be as long as 15, 16, 17, 18, 19 or 20 or more amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. Preferably, the salts are pharmaceutical acceptable salts of the peptides, such as, for example, the chloride or acetate (trifluoroacetate) salts. It has to be noted that the salts of the peptides according to the present invention differ substantially from the peptides in their state(s) in vivo, as the peptides are not salts in vivo.

The term “peptide” shall also include “oligopeptide”. The term “oligopeptide” is used herein to designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the oligopeptide is not critical to the invention, as long as the correct epitope or epitopes are maintained therein. The oligopeptides are typically less than about 30 amino acid residues in length, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the polypeptide is not critical to the invention as long as the correct epitopes are maintained. In contrast to the terms peptide or oligopeptide, the term polypeptide is meant to refer to molecules containing more than about 30 amino acid residues.

The term “full length polypeptide” means a complete protein (amino acid chain) or complete subunit (amino acid chain) of a multimeric protein.

A peptide, oligopeptide, protein or polynucleotide coding for such a molecule is “immunogenic” (and thus is an “immunogen” within the present invention), if it is capable of inducing an immune response. In the case of the present invention, immunogenicity is more specifically defined as the ability to induce a T-cell response. Thus, an “immunogen” would be a molecule that is capable of inducing an immune response, and in the case of the present invention, a molecule capable of inducing a T-cell response. In another aspect, the immunogen can be the peptide, the complex of the peptide with MHC, oligopeptide, and/or protein that is used to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to a class I MHC receptor, forming a ternary complex (MHC class I alpha chain, beta-2-microglobulin, and peptide) that can be recognized by a T cell bearing a matching T-cell receptor binding to the MHC/peptide complex with appropriate affinity. Peptides binding to MHC class I molecules are typically 8-14 amino acids in length, and most typically 9 amino acids in length.

In humans, there are three different genetic loci that encode MHC class I molecules (the MHC-molecules of the human are also designated human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the most frequent HLA-DR serotypes. Frequencies are deduced from haplotype frequencies Gf within the American population adapted from Mori et al. (Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 − Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might be enriched or less frequent than expected from their single frequencies due to linkage disequilibrium. For details refer to Chanock et al. (Chanock et al., 2004). Calculated phenotype from Allele Population allele frequency A*02 Caucasian (North America)  49.1% A*02 African American (North America)  34.1% A*02 Asian American (North America)  43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (North America)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian (North America)  20.6% DR4 Caucasian (North America)  30.7% DR5 Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7% DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7% DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20% DR2 African (North) American 29.80% DR3 African (North) American 24.80% DR4 African (North) American 11.10% DR5 African (North) American 31.10% DR6 African (North) American 33.70% DR7 African (North) American 19.20% DR8 African (North) American 12.10% DR9 African (North) American  5.80% DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3 Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5 Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7 Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9 Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2 Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4 Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6 Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8 Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24 Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24 Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 South Korea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29% A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24 South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccine of the invention as described herein bind to A*02. A vaccine may also include pan-binding MHC class II peptides. Therefore, the vaccine of the invention can be used to treat cancer in patients that are A*02 positive, whereas no selection for MHC class II allotypes is necessary due to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding to another allele, for example A*24, a higher percentage of any patient population can be treated compared with addressing either MHC class I allele alone. While in most populations less than 50% of patients could be addressed by either allele alone, a vaccine comprising HLA-A*24 and HLA-A*02 epitopes can treat at least 60% of patients in any relevant population. Specifically, the following percentages of patients will be positive for at least one of these alleles in various regions: USA 61%, Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculated from www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide, or polypeptide may be naturally occurring or they may be synthetically constructed. Generally, DNA segments encoding the peptides, polypeptides, and proteins of this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene that is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) a peptide” refers to a nucleotide sequence coding for the peptide including artificial (man-made) start and stop codons compatible for the biological system the sequence is to be expressed by, for example, a dendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes both single stranded and double stranded nucleic acid. Thus, for example for DNA, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.

The term “coding region” refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutated or altered gene, or can even be derived from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region, whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, by using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non-translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be paired with one strand of DNA and provides a free 3′-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNA polymerase to initiate transcription.

The term “isolated” means that the material is removed from its original environment (e.g., the natural environment, if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides, disclosed in accordance with the present invention may also be in “purified” form. The term “purified” does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by those of skill in the relevant art. For example, individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Furthermore, a claimed polypeptide which has a purity of preferably 99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weight or greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosed according to the present invention, as well as expression vectors containing such nucleic acids and/or such polypeptides, may be in “enriched form”. As used herein, the term “enriched” means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form. The term “active fragment” means a fragment, usually of a peptide, polypeptide or nucleic acid sequence, that generates an immune response (i.e., has immunogenic activity) when administered, alone or optionally with a suitable adjuvant or in a vector, to an animal, such as a mammal, for example, a rabbit or a mouse, and also including a human, such immune response taking the form of stimulating a T-cell response within the recipient animal, such as a human. Alternatively, the “active fragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide. When used in relation to polynucleotides, these terms refer to the products produced by treatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or “percent identical”, when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the “Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”). The percent identity is then determined according to the following formula: percent identity=100[1−(C/R)] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein

-   (i) each base or amino acid in the Reference Sequence that does not     have a corresponding aligned base or amino acid in the Compared     Sequence and -   (ii) each gap in the Reference Sequence and -   (iii) each aligned base or amino acid in the Reference Sequence that     is different from an aligned base or amino acid in the Compared     Sequence, constitutes a difference and -   (iiii) the alignment has to start at position 1 of the aligned     sequences;     and R is the number of bases or amino acids in the Reference     Sequence over the length of the alignment with the Compared Sequence     with any gap created in the Reference Sequence also being counted as     a base or amino acid.

If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.

As mentioned above, the present invention thus provides a peptide comprising a sequence that is selected from the group of consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 385, or a variant thereof that will induce T cells cross-reacting with said peptide. The peptides of the invention have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I or elongated versions of said peptides to class II.

In the present invention, the term “homologous” refers to the degree of identity (see percent identity above) between sequences of two amino acid sequences, i.e. peptide or polypeptide sequences. The aforementioned “homology” is determined by comparing two sequences aligned under optimal conditions over the sequences to be compared. Such a sequence homology can be calculated by creating an alignment using, for example, the ClustalW algorithm. Commonly available sequence analysis software, more specifically, Vector NTI, GENETYX or other tools are provided by public databases.

A person skilled in the art will be able to assess, whether T cells induced by a variant of a specific peptide will be able to cross-react with the peptide itself (Appay et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean that the side chains of, for example, one or two of the amino acid residues are altered (for example by replacing them with the side chain of another naturally occurring amino acid residue or some other side chain) such that the peptide is still able to bind to an HLA molecule in substantially the same way as a peptide consisting of the given amino acid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 385. For example, a peptide may be modified so that it at least maintains, if not improves, the ability to interact with and bind to the binding groove of a suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it at least maintains, if not improves, the ability to bind to the TCR of activated T cells.

These T cells can subsequently cross-react with cells and kill cells that express a polypeptide that contains the natural amino acid sequence of the cognate peptide as defined in the aspects of the invention. As can be derived from the scientific literature and databases (Rammensee et al., 1999; Godkin et al., 1997), certain positions of HLA binding peptides are typically anchor residues forming a core sequence fitting to the binding motif of the HLA receptor, which is defined by polar, electrophysical, hydrophobic and spatial properties of the polypeptide chains constituting the binding groove. Thus, one skilled in the art would be able to modify the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO 385, by maintaining the known anchor residues, and would be able to determine whether such variants maintain the ability to bind MHC class I or II molecules. The variants of the present invention retain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with and kill cells that express a polypeptide containing the natural amino acid sequence of the cognate peptide as defined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modified by the substitution of one or more residues at different, possibly selective, sites within the peptide chain, if not otherwise stated. Preferably those substitutions are located at the end of the amino acid chain. Such substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine. In studies of sequence variations in families of naturally occurring homologous proteins, certain amino acid substitutions are more often tolerated than others, and these are often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, and such is the basis for defining “conservative substitutions.”

Conservative substitutions are herein defined as exchanges within one of the following five groups: Group 1-small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar, positively charged residues (His, Arg, Lys); Group 4-large, aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of one amino acid by another that has similar characteristics but is somewhat different in size, such as replacement of an alanine by an isoleucine residue. Highly non-conservative replacements might involve substituting an acidic amino acid for one that is polar, or even for one that is basic in character. Such “radical” substitutions cannot, however, be dismissed as potentially ineffective since chemical effects are not totally predictable and radical substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles.

Of course, such substitutions may involve structures other than the common L-amino acids. Thus, D-amino acids might be substituted for the L-amino acids commonly found in the antigenic peptides of the invention and yet still be encompassed by the disclosure herein. In addition, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

If substitutions at more than one position are found to result in a peptide with substantially equivalent or greater antigenic activity as defined below, then combinations of those substitutions will be tested to determine if the combined substitutions result in additive or synergistic effects on the antigenicity of the peptide. At most, no more than 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicated herein can have one or two non-anchor amino acids (see below regarding the anchor motif) exchanged without that the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I or -II is substantially changed or is negatively affected, when compared to the non-modified peptide. In another embodiment, in a peptide consisting essentially of the amino acid sequence as indicated herein, one or two amino acids can be exchanged with their conservative exchange partners (see herein below) without that the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I or -II is substantially changed, or is negatively affected, when compared to the non-modified peptide.

The amino acid residues that do not substantially contribute to interactions with the T-cell receptor can be modified by replacement with other amino acids whose incorporation does not substantially affect T-cell reactivity and does not eliminate binding to the relevant MHC. Thus, apart from the proviso given, the peptide of the invention may be any peptide (by which term the inventors include oligopeptide or polypeptide), which includes the amino acid sequences or a portion or variant thereof as given.

TABLE 6  Variants and motif of the peptides according to SEQ ID NO: 2, 5, 8, 62, and 53 Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 2 A L H R P D V Y L Variants V I A M M V M I M A A A V A I A A V V V V I V A T T V T I T A Q Q V Q I Q A SEQ ID NO 5 A L I F K I A S A Variants L I V M M L M I M V A A L A I A V V V L V I V V T T L T I T V Q Q L Q I Q V SEQ ID NO. 8 A L A A S A L P A L V Variants I L A M M I M L M A A A I A L A A V V I V L V A T T I T L T A Q Q I Q L Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID 62 G I I D G S P R L Variants L V L I L L A M V M I M M A A V A I A A A V V V I V V A T V T I T T A Q V Q I Q Q A SEQ ID 153 S L L A E L H V L Variants V I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A Q V Q I Q Q A

Longer (elongated) peptides may also be suitable. It is possible that MHC class I epitopes, although usually between 8 and 11 amino acids long, are generated by peptide processing from longer peptides or proteins that include the actual epitope. It is preferred that the residues that flank the actual epitope are residues that do not substantially affect proteolytic cleavage necessary to expose the actual epitope during processing.

The peptides of the invention can be elongated by up to four amino acids, that is 1, 2, 3 or 4 amino acids can be added to either end in any combination between 4:0 and 0:4. Combinations of the elongations according to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the invention C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of the original sequence of the protein or any other amino acid(s). The elongation can be used to enhance the stability or solubility of the peptides.

Thus, the epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes or may include epitopes that differ by no more than four residues from the reference peptide, as long as they have substantially identical antigenic activity.

In an alternative embodiment, the peptide is elongated on either or both sides by more than 4 amino acids, preferably to a total length of up to 30 amino acids. This may lead to MHC class II binding peptides. Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHC class I epitopes, wherein the peptide or variant has an overall length of between 8 and 100, preferably between 8 and 30, and most preferred between 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in case of the elongated class II binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present invention will have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class I or II. Binding of a peptide or a variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to the present invention are tested against the substituted peptides, the peptide concentration at which the substituted peptides achieve half the maximal increase in lysis relative to background is no more than about 1 mM, preferably no more than about 1 μM, more preferably no more than about 1 nM, and still more preferably no more than about 100 pM, and most preferably no more than about 10 pM. It is also preferred that the substituted peptide be recognized by T cells from more than one individual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptide consists or consists essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 385.

“Consisting essentially of” shall mean that a peptide according to the present invention, in addition to the sequence according to any of SEQ ID NO: 1 to SEQ ID NO 385 or a variant thereof contains additional N- and/or C-terminally located stretches of amino acids that are not necessarily forming part of the peptide that functions as an epitope for MHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficient introduction of the peptide according to the present invention into the cells. In one embodiment of the present invention, the peptide is part of a fusion protein which comprises, for example, the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, in the following “Ii”) as derived from the NCBI, GenBank Accession number X00497. In other fusions, the peptides of the present invention can be fused to an antibody as described herein, or a functional part thereof, in particular into a sequence of an antibody, so as to be specifically targeted by said antibody, or, for example, to or into an antibody that is specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improve stability and/or binding to MHC molecules in order to elicit a stronger immune response. Methods for such an optimization of a peptide sequence are well known in the art and include, for example, the introduction of reverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide (—CO—NH—) linkages but the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) (Meziere et al., 1997), incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al. (Meziere et al., 1997) show that for MHC binding and T helper cell responses, these pseudopeptides are useful. Retro-inverse peptides, which contain NH—CO bonds instead of CO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—, —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides a method for the solid phase synthesis of non-peptide bonds (—CH₂—NH) in polypeptide chains which involves polypeptides synthesized by standard procedures and the non-peptide bond synthesized by reacting an amino aldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, to enhance the stability, bioavailability, and/or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides' amino termini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter their steric configuration. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used, rather than the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well-known non-naturally occurring amino acid residues. Alterations such as these may serve to increase the stability, bioavailability and/or binding action of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modified chemically by reacting specific amino acids either before or after synthesis of the peptide. Examples for such modifications are well known in the art and are summarized e.g. in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which is incorporated herein by reference. Chemical modification of amino acids includes but is not limited to, modification by acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by performic acid oxidation of cysteine to cysteic acid, formation of mercurial derivatives, formation of mixed disulphides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoylation with cyanate at alkaline pH, although without limitation thereto. In this regard, the skilled person is referred to Chapter 15 of Current Protocols In Protein Science, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995) for more extensive methodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form an adduct. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites such as lysine and histidine. As a result, a large number of reagents are available for the modification of cysteine. The websites of companies such as Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Woodward's Reagent K may be used to modify specific glutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be used to form intra-molecular crosslinks between a lysine residue and a glutamic acid residue. For example, diethylpyrocarbonate is a reagent for the modification of histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. The reaction of lysine residues and other α-amino groups is, for example, useful in binding of peptides to surfaces or the cross-linking of proteins/peptides. Lysine is the site of attachment of poly(ethylene)glycol and the major site of modification in the glycosylation of proteins. Methionine residues in proteins can be modified with e.g. iodoacetamide, bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl residues. Cross-linking via the formation of dityrosine can be accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG is often associated with an extension of circulatory half-life while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includes non-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturally occurring peptide wherein said peptide consists or consists essentially of an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 385 and has been synthetically produced (e.g. synthesized) as a pharmaceutically acceptable salt. Methods to synthetically produce peptides are well known in the art. The salts of the peptides according to the present invention differ substantially from the peptides in their state(s) in vivo, as the peptides as generated in vivo are no salts. The non-natural salt form of the peptide mediates the solubility of the peptide, in particular in the context of pharmaceutical compositions comprising the peptides, e.g. the peptide vaccines as disclosed herein. A sufficient and at least substantial solubility of the peptide(s) is required in order to efficiently provide the peptides to the subject to be treated. Preferably, the salts are pharmaceutically acceptable salts of the peptides. These salts according to the invention include alkaline and earth alkaline salts such as salts of the Hofmeister series comprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄, (NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄, CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr, LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, and Ba(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate (trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptide linkages between amino acid residues) may be synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lukas et al. (Lukas et al., 1981) and by references as cited therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4′-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/1 hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used include ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. Also a combination of solid phase and solution phase methodologies for the synthesis of peptides is possible (see, for example, (Bruckdorfer et al., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilization of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from e.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of, techniques such as re-crystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography and (usually) reverse-phase high performance liquid chromatography using e.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.

In order to select over-presented peptides, a presentation profile is calculated showing the median sample presentation as well as replicate variation. The profile juxtaposes samples of the tumor entity of interest to a baseline of normal tissue samples. Each of these profiles can then be consolidated into an over-presentation score by calculating the p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015) adjusting for multiple testing by False Discovery Rate (Benjamini and Hochberg, 1995) (cf. Example 1, FIG. 1).

For the identification and relative quantitation of HLA ligands by mass spectrometry, HLA molecules from shock-frozen tissue samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated and sequences were identified by online nano-electrospray-ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were verified by comparison of the fragmentation pattern of natural tumor-associated peptides (TUMAPs) recorded from CLL samples (N=17 A*02-positive samples) with the fragmentation patterns of corresponding synthetic reference peptides of identical sequences. Since the peptides were directly identified as ligands of HLA molecules of primary tumors, these results provide direct evidence for the natural processing and presentation of the identified peptides on primary cancer tissue obtained from 16 CLL patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US 2013-0096016, which is hereby incorporated by reference in its entirety) allows the identification and selection of relevant over-presented peptide vaccine candidates based on direct relative quantitation of HLA-restricted peptide levels on cancer tissues in comparison to several different non-cancerous tissues and organs. This was achieved by the development of label-free differential quantitation using the acquired LC-MS data processed by a proprietary data analysis pipeline, combining algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, charge state deconvolution and normalization.

Presentation levels including error estimates for each peptide and sample were established. Peptides exclusively presented on tumor tissue and peptides over-presented in tumor versus non-cancerous tissues and organs have been identified.

HLA-peptide complexes from CLL tissue samples were purified and HLA-associated peptides were isolated and analyzed by LC-MS (see examples). All TUMAPs contained in the present application were identified with this approach on primary CLL samples confirming their presentation on primary CLL.

TUMAPs identified on multiple CLL and normal tissues were quantified using ion-counting of label-free LC-MS data. The method assumes that LC-MS signal areas of a peptide correlate with its abundance in the sample. All quantitative signals of a peptide in various LC-MS experiments were normalized based on central tendency, averaged per sample and merged into a bar plot, called presentation profile. The presentation profile consolidates different analysis methods like protein database search, spectral clustering, charge state deconvolution (decharging) and retention time alignment and normalization.

Furthermore, the discovery platform Technique XPRESIDENT® v2.x (see for example PCT/EP2011/056056 and PCT/EP2015/079873) allows the direct absolute quantitation of MHC-, preferably HLA-restricted, peptide levels on cancer or other infected tissues. Briefly, the total cell count was calculated from the total DNA content of the analyzed tissue sample. The total peptide amount for a TUMAP in a tissue sample was measured by nanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of an isotope-labelled version of the TUMAP, the so-called internal standard. The efficiency of TUMAP isolation was determined by spiking peptide:MHC complexes of all selected TUMAPs into the tissue lysate at the earliest possible point of the TUMAP isolation procedure and their detection by nanoLC-MS/MS following completion of the peptide isolation procedure. The total cell count and the amount of total peptide were calculated from triplicate measurements per tissue sample. The peptide-specific isolation efficiencies were calculated as an average from 10 spike experiments each measured as a triplicate (see Example 6 and Table 12).

Besides over-presentation of the peptide, mRNA expression of the underlying gene was tested. mRNA data were obtained via RNASeq analyses of normal tissues and cancer tissues (cf. Example 2, FIG. 3). An additional source of normal tissue data was a database of publicly available RNA expression data from around 3000 normal tissue samples (Lonsdale, 2013). Peptides which are derived from proteins whose coding mRNA is highly expressed in cancer tissue, but very low or absent in vital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treating cancers/tumors, preferably CLL that over- or exclusively present the peptides of the invention. These peptides were shown by mass spectrometry to be naturally presented by HLA molecules on primary human CLL samples.

Many of the source gene/proteins (also designated “full-length proteins” or “underlying proteins”) from which the peptides are derived were shown to be highly over-expressed in cancer compared with normal tissues—“normal tissues” in relation to this invention shall mean either healthy adipose tissue, adrenal gland, blood cells, blood vessel, bone marrow, brain, breast, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter, and urinary bladder cells or other normal tissue cells, demonstrating a high degree of tumor association of the source genes (see Example 2). Moreover, the peptides themselves are strongly over-presented on tumor tissue—“tumor tissue” in relation to this invention shall mean a sample from a patient suffering from CLL, but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specifically T lymphocytes. T cells can destroy the cells presenting the recognized HLA/peptide complex, e.g. CLL cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable of stimulating T cell responses and/or are over-presented and thus can be used for the production of antibodies and/or TCRs, such as soluble TCRs, according to the present invention (see Example 3, Example 4). Furthermore, the peptides when complexed with the respective MHC can be used for the production of antibodies and/or TCRs, in particular sTCRs, according to the present invention, as well. Respective methods are well known to the person of skill, and can be found in the respective literature as well. Thus, the peptides of the present invention are useful for generating an immune response in a patient by which tumor cells can be destroyed. An immune response in a patient can be induced by direct administration of the described peptides or suitable precursor substances (e.g. elongated peptides, proteins, or nucleic acids encoding these peptides) to the patient, ideally in combination with an agent enhancing the immunogenicity (i.e. an adjuvant). The immune response originating from such a therapeutic vaccination can be expected to be highly specific against tumor cells because the target peptides of the present invention are not presented on normal tissues in comparable copy numbers, preventing the risk of undesired autoimmune reactions against normal cells in the patient.

The present description further relates to T-cell receptors (TCRs) comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Also provided are peptides according to the invention capable of binding to TCRs and antibodies when presented by an MHC molecule. The present description also relates to nucleic acids, vectors and host cells for expressing TCRs and peptides of the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), wherein the heterodimeric receptor is capable of binding to a peptide antigen presented by an HLA molecule. The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCR as described herein, the method comprising culturing a host cell capable of expressing the TCR under conditions suitable to promote expression of the TCR.

The description in another aspect relates to methods according to the description, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting a sufficient amount of the antigen with an antigen-presenting cell or the antigen is loaded onto class I or II MHC tetramers by tetramerizing the antigen/class I or II MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and delta chains of gamma/delta TCRs, are generally regarded as each having two “domains”, namely variable and constant domains. The variable domain consists of a concatenation of variable region (V), and joining region (J). The variable domain may also include a leader region (L). Beta and delta chains may also include a diversity region (D). The alpha and beta constant domains may also include C-terminal transmembrane (TM) domains that anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain” as used herein refers to the concatenation of the TCR gamma V (TRGV) region without leader region (L), and the TCR gamma J (TRGJ) region, and the term TCR gamma constant domain refers to the extracellular TRGC region, or to a C-terminal truncated TRGC sequence. Likewise the term “TCR delta variable domain” refers to the concatenation of the TCR delta V (TRDV) region without leader region (L) and the TCR delta D/J (TRDD/TRDJ) region, and the term “TCR delta constant domain” refers to the extracellular TRDC region, or to a C-terminal truncated TRDC sequence.

TCRs of the present description preferably bind to an peptide-HLA molecule complex with a binding affinity (KD) of about 100 μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM or less. More preferred are high affinity TCRs having binding affinities of about 1 μM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less. Non-limiting examples of preferred binding affinity ranges for TCRs of the present invention include about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description, “specific binding” and grammatical variants thereof are used to mean a TCR having a binding affinity (KD) for a peptide-HLA molecule complex of 100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have an introduced disulfide bond between their constant domains. Preferred TCRs of this type include those which have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the said cysteines forming a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above, alpha/beta heterodimeric TCRs of the present description may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selected from the group consisting of a radionuclide, a fluorophore and biotin. TCRs of the present description may be conjugated to a therapeutically active agent, such as a radionuclide, a chemotherapeutic agent, or a toxin.

In an embodiment, a TCR of the present description having at least one mutation in the alpha chain and/or having at least one mutation in the beta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCR alpha chain and/or TCR beta chain has a binding affinity for, and/or a binding half-life for, a peptide-HLA molecule complex, which is at least double that of a TCR comprising the unmutated TCR alpha chain and/or unmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs, and its exploitation, relies on the existence of a window for optimal TCR affinities. The existence of such a window is based on observations that TCRs specific for HLA-A2-restricted pathogens have KD values that are generally about 10-fold lower when compared to TCRs specific for HLA-A2-restricted tumor-associated self-antigens. It is now known, although tumor antigens have the potential to be immunogenic, because tumors arise from the individual's own cells only mutated proteins or proteins with altered translational processing will be seen as foreign by the immune system. Antigens that are upregulated or overexpressed (so called self-antigens) will not necessarily induce a functional immune response against the tumor: T-cells expressing TCRs that are highly reactive to these antigens will have been negatively selected within the thymus in a process known as central tolerance, meaning that only T-cells with low-affinity TCRs for self-antigens remain. Therefore, affinity of TCRs or variants of the present description to peptides can be enhanced by methods well known in the art.

The present description further relates to a method of identifying and isolating a TCR according to the present description, said method comprising incubating PBMCs from HLA-A*02-negative healthy donors with A2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin (PE) and isolating the high avidity T-cells by fluorescence activated cell sorting (FACS)—Calibur analysis.

The present description further relates to a method of identifying and isolating a TCR according to the present description, said method comprising obtaining a transgenic mouse with the entire human TCRaβ gene loci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency, immunizing the mouse with a peptide, incubating PBMCs obtained from the transgenic mice with tetramer-phycoerythrin (PE), and isolating the high avidity T-cells by fluorescence activated cell sorting (FACS)—Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the present description, nucleic acids encoding TCR-alpha and/or TCR-beta chains of the present description are cloned into expression vectors, such as gamma retrovirus or lentivirus. The recombinant viruses are generated and then tested for functionality, such as antigen specificity and functional avidity. An aliquot of the final product is then used to transduce the target T-cell population (generally purified from patient PBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the present description, TCR RNAs are synthesized by techniques known in the art, e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAs are then introduced into primary CD8+ T-cells obtained from healthy donors by electroporation to re-express tumor specific TCR-alpha and/or TCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the present description may be operably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin, ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter, elongation factor (EF)-1a and the spleen focus-forming virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the nucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the present description may contain additional elements that can enhance transgene expression, including a central polypurine tract (cPPT), which promotes the nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE), which increases the level of transgene expression by increasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may be encoded by nucleic acids located in separate vectors, or may be encoded by polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both the TCR-alpha and TCR-beta chains of the introduced TCR be transcribed at high levels. To do so, the TCR-alpha and TCR-beta chains of the present description may be cloned into bi-cistronic constructs in a single vector, which has been shown to be capable of over-coming this obstacle. The use of a viral intraribosomal entry site (IRES) between the TCR-alpha and TCR-beta chains results in the coordinated expression of both chains, because the TCR-alpha and TCR-beta chains are generated from a single transcript that is broken into two proteins during translation, ensuring that an equal molar ratio of TCR-alpha and TCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codon optimized to increase expression from a host cell. Redundancy in the genetic code allows some amino acids to be encoded by more than one codon, but certain codons are less “op-timal” than others because of the relative availability of matching tRNAs as well as other factors (Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta gene sequences such that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as eliminating mRNA instability motifs or cryptic splice sites, has been shown to significantly enhance TCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chains may result in the acquisition of specificities that pose a significant risk for autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form properly paired TCR complexes, and therefore can significantly decrease the functional avidity of the cells expressing the introduced TCR (Kuball et al., 2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chains of the present description may be modified in order to promote interchain affinity, while de-creasing the ability of the introduced chains to pair with the endogenous TCR. These strategies may include replacing the human TCR-alpha and TCR-beta C-terminus domains with their murine counterparts (murinized C-terminus domain); generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both the TCR-alpha and TCR-beta chains of the introduced TCR (cysteine modification); swapping interacting residues in the TCR-alpha and TCR-beta chain C-terminus domains (“knob-in-hole”); and fusing the variable domains of the TCR-alpha and TCR-beta chains directly to CD3ξ (CD3ξ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of the present description. In preferred embodiments, the host cell is a human T-cell or T-cell progenitor. In some embodiments the T-cell or T-cell progenitor is obtained from a cancer patient. In other embodiments the T-cell or T-cell progenitor is obtained from a healthy donor. Host cells of the present description can be allogeneic or autologous with respect to a patient to be treated. In one embodiment, the host is a gamma/delta T-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable for administration to a human being in a medical setting. Preferably, a pharmaceutical composition is sterile and produced according to GMP guidelines.

The pharmaceutical compositions comprise the peptides either in the free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, “a pharmaceutically acceptable salt” refers to a derivative of the disclosed peptides wherein the peptide is modified by making acid or base salts of the agent. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral—NH2 group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like. Conversely, preparation of basic salts of acid moieties which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.

In an especially preferred embodiment, the pharmaceutical compositions comprise the peptides as salts of acetic acid (acetates), trifluoro acetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is an immunotherapeutic such as a vaccine. It may be administered directly into the patient, into the affected organ or systemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo to cells derived from the patient or a human cell line which are subsequently administered to the patient, or used in vitro to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. If the nucleic acid is administered to cells in vitro, it may be useful for the cells to be transfected so as to co-express immune-stimulating cytokines, such as interleukin-2. The peptide may be substantially pure, or combined with an immune-stimulating adjuvant (see below) or used in combination with immune-stimulatory cytokines, or be administered with a suitable delivery system, for example liposomes. The peptide may also be conjugated to a suitable carrier such as keyhole limpet haemocyanin (KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). The peptide may also be tagged, may be a fusion protein, or may be a hybrid molecule. The peptides whose sequence is given in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help provided by CD4 T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 T cells the fusion partner or sections of a hybrid molecule suitably provide epitopes which stimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes are well known in the art and include those identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having the amino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 385, and at least one additional peptide, preferably two to 50, more preferably two to 25, even more preferably two to 20 and most preferably two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen peptides. The peptide(s) may be derived from one or more specific TAAs and may bind to MHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example a polynucleotide) encoding a peptide or peptide variant of the invention. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA or combinations thereof, either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone and it may or may not contain introns so long as it codes for the peptide. Of course, only peptides that contain naturally occurring amino acid residues joined by naturally occurring peptide bonds are encodable by a polynucleotide. A still further aspect of the invention provides an expression vector capable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of the invention employs the polymerase chain reaction as disclosed by Saiki R K, et al. (Saiki et al., 1988). This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art. If viral vectors are used, pox- or adenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. Thus, the DNA encoding the peptide or variant of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.

Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus spec.), plant cells, animal cells and insect cells. Preferably, the system can be mammalian cells such as CHO cells available from the ATCC Cell Biology Collection.

A typical mammalian cell vector plasmid for constitutive expression comprises the CMV or SV40 promoter with a suitable poly A tail and a resistance marker, such as neomycin. One example is pSVL available from Pharmacia, Piscataway, N.J., USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps). CMV promoter-based vectors (for example from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in various combinations of FLAG, 3×FLAG, c-myc or MAT. These fusion proteins allow for detection, purification and analysis of recombinant protein. Dual-tagged fusions provide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg/L in COS cells. For less potent cell lines, protein levels are typically ˜0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive COS cells. CMV vectors, for example, can contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the f1 origin. Vectors containing the pre-pro-trypsin leader (PPT) sequence can direct the secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG antibodies, resins, and plates. Other vectors and expression systems are well known in the art for use with a variety of host cells.

In another embodiment two or more peptides or peptide variants of the invention are encoded and thus expressed in a successive order (similar to “beads on a string” constructs). In doing so, the peptides or peptide variants may be linked or fused together by stretches of linker amino acids, such as for example LLLLLL, or may be linked without any additional peptide(s) between them. These constructs can also be used for cancer therapy, and may induce immune responses both involving MHC I and MHC II.

The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some circumstances and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and colon cell lines. Yeast host cells include YPH499, YPH500 and YPH501, which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293 cells which are human embryonic kidney cells. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors. An overview regarding the choice of suitable host cells for expression can be found in, for example, the textbook of Paulina Balbás and Argelia Lorence “Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols,” Part One, Second Edition, ISBN 978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well-known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). Transformation of yeast cells is described in Sherman et al. (Sherman et al., 1986). The method of Beggs (Beggs, 1978) is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA. Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA construct of the present invention, can be identified by well-known techniques such as PCR. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention are useful in the preparation of the peptides of the invention, for example bacterial, yeast and insect cells. However, other host cells may be useful in certain therapeutic methods. For example, antigen-presenting cells, such as dendritic cells, may usefully be used to express the peptides of the invention such that they may be loaded into appropriate MHC molecules. Thus, the current invention provides a host cell comprising a nucleic acid or an expression vector according to the invention.

In a preferred embodiment, the host cell is an antigen presenting cell, in particular a dendritic cell or antigen presenting cell. APCs loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) were approved by the U.S. Food and Drug Administration (FDA) on Apr. 29, 2010, to treat asymptomatic or minimally symptomatic metastatic HRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing a peptide or its variant, the method comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expression vector of the invention are used in medicine. For example, the peptide or its variant may be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection include i.d., i.m., s.c., i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μg to 500 μg, of peptide or DNA may be given and will depend on the respective peptide or DNA. Dosages of this range were successfully used in previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantially pure, or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods for designing and introducing such a nucleic acid are well known in the art. An overview is provided by e.g. Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines are easy to prepare, but the mode of action of these vectors in inducing an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus or hybrids containing elements of more than one virus. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in the art of DNA delivery. Physical delivery, such as via a “gene-gun” may also be used. The peptide or peptides encoded by the nucleic acid may be a fusion protein, for example with an epitope that stimulates T cells for the respective opposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate the immune response (e.g., immune responses mediated by CD8-positive T cells and helper-T (TH) cells to an antigen, and would thus be considered useful in the medicament of the present invention. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactid co-glycolid) [PLG]-based and dextran microparticles, talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Allison and Krummel, 1995). Also cytokines may be used. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).

CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly it enhances dendritic cell maturation and differentiation, resulting in enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a TH2 bias. CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak. They also accelerate the immune response and enable the antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG in some experiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response. A CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany) which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited to chemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such as Poly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system (e.g. anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may act therapeutically and/or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and derivates, poly-(I:C) and derivates, RNA, sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according to the invention the adjuvant is selected from the group consisting of colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according to the invention the adjuvant is selected from the group consisting of colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinations thereof.

This composition is used for parenteral administration, such as subcutaneous, intradermal, intramuscular or oral administration. For this, the peptides and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. The peptides can also be administered together with immune stimulating substances, such as cytokines. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for a prevention, prophylaxis and/or therapy of adenomatous or cancerous diseases. Exemplary formulations can be found in, for example, EP2112253.

It is important to realize that the immune response triggered by the vaccine according to the invention attacks the cancer in different cell-stages and different stages of development. Furthermore, different cancer associated signaling pathways are attacked. This is an advantage over vaccines that address only one or few targets, which may cause the tumor to easily adapt to the attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, a combination of several tumor-associated peptides ensures that every single tumor bears at least some of the targets. The composition is designed in such a way that each tumor is expected to express several of the antigens and cover several independent pathways necessary for tumor growth and maintenance. Thus, the vaccine can easily be used “off-the-shelf” for a larger patient population. This means that a pre-selection of patients to be treated with the vaccine can be restricted to HLA typing, does not require any additional biomarker assessments for antigen expression, but it is still ensured that several targets are simultaneously attacked by the induced immune response, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

As used herein, the term “scaffold” refers to a molecule that specifically binds to an (e.g. antigenic) determinant. In one embodiment, a scaffold is able to direct the entity to which it is attached (e.g. a (second) antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant (e.g. the complex of a peptide with MHC, according to the application at hand). In another embodiment, a scaffold is able to activate signaling through its target antigen, for example a T cell receptor complex antigen. Scaffolds include but are not limited to antibodies and fragments thereof, antigen binding domains of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region, binding proteins comprising at least one ankyrin repeat motif and single domain antigen binding (SDAB) molecules, aptamers, (soluble) TCRs and (modified) cells such as allogenic or autologous T cells. To assess whether a molecule is a scaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complex of interest better than other naturally occurring peptide-MHC-complexes, to an extent that a scaffold armed with an active molecule that is able to kill a cell bearing the specific target is not able to kill another cell without the specific target but presenting other peptide-MHC complex(es). Binding to other peptide-MHC complexes is irrelevant if the peptide of the cross-reactive peptide-MHC is not naturally occurring, i.e. not derived from the human HLA-peptidome. Tests to assess target cell killing are well known in the art. They should be performed using target cells (primary cells or cell lines) with unaltered peptide-MHC presentation, or cells loaded with peptides such that naturally occurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the bound scaffold can be detected by determining the presence or absence of a signal provided by the label. For example, the scaffold can be labelled with a fluorescent dye or any other applicable cellular marker molecule. Such marker molecules are well known in the art. For example, a fluorescence-labelling, for example provided by a fluorescence dye, can provide a visualization of the bound aptamer by fluorescence or laser scanning microscopy or flow cytometry. Each scaffold can be conjugated with a second active molecule such as for example IL-21, anti-CD3, anti-CD28. For further information on polypeptide scaffolds see for example the background section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see for example WO 2014/191359 and the literature as cited therein) are short single-stranded nucleic acid molecules, which can fold into defined three-dimensional structures and recognize specific target structures. They have appeared to be suitable alternatives for developing targeted therapies. Aptamers have been shown to selectively bind to a variety of complex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identified within the past decade and provide means for developing diagnostic and therapeutic approaches. Since aptamers have been shown to possess almost no toxicity and immunogenicity they are promising candidates for biomedical applications. Indeed, aptamers, for example prostate-specific membrane-antigen recognizing aptamers, have been successfully employed for targeted therapies and shown to be functional in xenograft in vivo models. Furthermore, aptamers recognizing specific tumor cell lines have been identified.

DNA aptamers can be selected to reveal broad-spectrum recognition properties for various cancer cells, and particularly those derived from solid tumors, while non-tumorigenic and primary healthy cells are not recognized. If the identified aptamers recognize not only a specific tumor sub-type but rather interact with a series of tumors, this renders the aptamers applicable as so-called broad-spectrum diagnostics and therapeutics.

Further, investigation of cell-binding behavior with flow cytometry showed that the aptamers revealed very good apparent affinities that are within the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, it could be shown that some of the aptamers are taken up by tumor cells and thus can function as molecular vehicles for the targeted delivery of anti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells and tissues and complexes of the peptides comprising, preferably consisting of, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 385, according to the invention at hand with the MHC molecule, using the cell-SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique.

The peptides of the present invention can be used to generate and develop specific antibodies against MHC/peptide complexes. These can be used for therapy, targeting toxins or radioactive substances to the diseased tissue. Another use of these antibodies can be targeting radionuclides to the diseased tissue for imaging purposes such as PET. This use can help to detect small metastases or to determine the size and precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a method for producing a recombinant antibody specifically binding to a human major histocompatibility complex (MHC) class I or II being complexed with a HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal comprising cells expressing said human major histocompatibility complex (MHC) class I or II with a soluble form of a MHC class I or II molecule being complexed with said HLA-restricted antigen; isolating mRNA molecules from antibody producing cells of said non-human mammal; producing a phage display library displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, said at least one phage displaying said antibody specifically binding to said human major histocompatibility complex (MHC) class I or II being complexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody that specifically binds to a human major histocompatibility complex (MHC) class I or II being complexed with a HLA-restricted antigen, wherein the antibody preferably is a polyclonal antibody, monoclonal antibody, bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain class I major histocompatibility complexes, as well as other tools for the production of these antibodies are disclosed in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for the purposes of the present invention are all explicitly incorporated by reference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20 nanomolar, preferably of below 10 nanomolar, to the complex, which is also regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385, or a variant thereof which is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof that induces T cells cross-reacting with said peptide, wherein said peptide is not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequence that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof which is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 385, wherein said peptide or variant has an overall length of between 8 and 100, preferably between 8 and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to the invention that have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to the invention wherein the peptide consists or consists essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 385.

The present invention further relates to the peptides according to the invention, wherein the peptide is (chemically) modified and/or includes non-peptide bonds.

The present invention further relates to the peptides according to the invention, wherein the peptide is part of a fusion protein, in particular comprising N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or wherein the peptide is fused to (or into) an antibody, such as, for example, an antibody that is specific for dendritic cells.

The present invention further relates to a nucleic acid, encoding the peptides according to the invention, provided that the peptide is not the complete (full) human protein.

The present invention further relates to the nucleic acid according to the invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable of expressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to the present invention, a nucleic acid according to the present invention or an expression vector according to the present invention for use in medicine, in particular in the treatment of CLL.

The present invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector according to the invention.

The present invention further relates to the host cell according to the present invention that is an antigen presenting cell, and preferably a dendritic cell.

The present invention further relates to a method of producing a peptide according to the present invention, said method comprising culturing the host cell according to the present invention, and isolating the peptide from said host cell or its culture medium.

The present invention further relates to the method according to the present invention, where-in the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell by contacting a sufficient amount of the antigen with an antigen-presenting cell.

The present invention further relates to the method according to the invention, wherein the antigen-presenting cell comprises an expression vector capable of expressing or expressing said peptide containing SEQ ID NO: 1 to SEQ ID NO: 385 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced by the method according to the present invention, wherein said T cells selectively recognizes a cell which aberrantly expresses a polypeptide comprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing target cells in a patient which target cells aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, the method comprising administering to the patient an effective number of T cells as according to the present invention.

The present invention further relates to the use of any peptide described, a nucleic acid according to the present invention, an expression vector according to the present invention, a cell according to the present invention, or an activated cytotoxic T lymphocyte according to the present invention as a medicament or in the manufacture of a medicament. The present invention further relates to a use according to the present invention, wherein the medicament is active against cancer.

The present invention further relates to a use according to the invention, wherein the medicament is a vaccine. The present invention further relates to a use according to the invention, wherein the medicament is active against cancer.

The present invention further relates to a use according to the invention, wherein said cancer cells are CLL cells or other solid or hematological tumor cells such as acute myelogenous leukemia, bile duct cancer, brain cancer, breast cancer, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, urinary bladder cancer and uterine cancer.

The present invention further relates to particular marker proteins and biomarkers based on the peptides according to the present invention, herein called “targets” that can be used in the diagnosis and/or prognosis of CLL. The present invention also relates to the use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact or “full” immunoglobulin molecules, also included in the term “antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties (e.g., specific binding of a CLL marker (poly)peptide, delivery of a toxin to a CLL cell expressing a cancer marker gene at an increased level, and/or inhibiting the activity of a CLL marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased from commercial sources. The antibodies of the invention may also be generated using well-known methods. The skilled artisan will understand that either full length CLL marker polypeptides or fragments thereof may be used to generate the antibodies of the invention. A polypeptide to be used for generating an antibody of the invention may be partially or fully purified from a natural source, or may be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the present invention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 385 polypeptide, or a variant or fragment thereof, can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells), after which the recombinant protein can be purified and used to generate a monoclonal or polyclonal antibody preparation that specifically bind the CLL marker polypeptide used to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining an antibody with the specificity and affinity required for its intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). The antibodies are tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on the generation and testing of antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). For example, the antibodies may be tested in ELISA assays or, Western blots, immunohistochemical staining of formalin-fixed cancers or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. Pat. No. 4,816,567, which is hereby incorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridoma methods. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. Human antibodies can also be produced in phage display libraries.

Antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered.

The antibodies can be administered to the subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. The antibodies may also be administered by intratumoral or peritumoral routes, to exert local as well as systemic therapeutic effects. Local or intravenous injection is preferred.

Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered. A typical daily dosage of the antibody used alone might range from about 1 (μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. Following administration of an antibody, preferably for treating CLL, the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled practitioner. For instance, the size, number, and/or distribution of cancer in a subject receiving treatment may be monitored using standard tumor imaging techniques. A therapeutically-administered antibody that arrests tumor growth, results in tumor shrinkage, and/or prevents the development of new tumors, compared to the disease course that would occurs in the absence of antibody administration, is an efficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method for producing a soluble T-cell receptor (sTCR) recognizing a specific peptide-MHC complex. Such soluble T-cell receptors can be generated from specific T-cell clones, and their affinity can be increased by mutagenesis targeting the complementarity-determining regions. For the purpose of T-cell receptor selection, phage display can be used (US 2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization of T-cell receptors during phage display and in case of practical use as drug, alpha and beta chain can be linked e.g. by non-native disulfide bonds, other covalent bonds (single-chain T-cell receptor), or by dimerization domains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). The T-cell receptor can be linked to toxins, drugs, cytokines (see, for example, US 2013/0115191), domains recruiting effector cells such as an anti-CD3 domain, etc., in order to execute particular functions on target cells. Moreover, it could be expressed in T cells used for adoptive transfer. Further information can be found in WO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs is described in WO 2012/056407A1. Further methods for the production are disclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other binding molecules of the present invention can be used to verify a pathologist's diagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionucleotide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can be localized using immunoscintiography. In one embodiment, antibodies or fragments thereof bind to the extracellular domains of two or more targets of a protein selected from the group consisting of the above-mentioned proteins, and the affinity value (Kd) is less than 1×10 μM.

Antibodies for diagnostic use may be labeled with probes suitable for detection by various imaging methods. Methods for detection of probes include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides. Additionally, probes may be bi- or multi-functional and be detectable by more than one of the methods listed. These antibodies may be directly or indirectly labeled with said probes. Attachment of probes to the antibodies includes covalent attachment of the probe, incorporation of the probe into the antibody, and the covalent attachment of a chelating compound for binding of probe, amongst others well recognized in the art. For immunohistochemistry, the disease tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin. The fixed or embedded section contains the sample are contacted with a labeled primary antibody and secondary antibody, wherein the antibody is used to detect the expression of the proteins in situ.

Another aspect of the present invention includes an in vitro method for producing activated T cells, the method comprising contacting in vitro T cells with antigen loaded human MHC molecules expressed on the surface of a suitable antigen-presenting cell for a period of time sufficient to activate the T cell in an antigen specific manner, wherein the antigen is a peptide according to the invention. Preferably a sufficient amount of the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or function of the TAP peptide transporter. Suitable cells that lack the TAP peptide transporter include T2, RMA-S and Drosophila cells. TAP is the transporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA under Catalogue No CRL 1992; the Drosophila cell line Schneider line 2 is available from the ATCC under Catalogue No CRL 19863; the mouse RMA-S cell line is described in Ljunggren et al. (Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially no MHC class I molecules. It is also preferred that the stimulator cell expresses a molecule important for providing a co-stimulatory signal for T-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acid sequences of numerous MHC class I molecules and of the co-stimulator molecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cells are CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope, preferably the cell comprises an expression vector capable of expressing a peptide containing SEQ ID NO: 1 to SEQ ID NO: 385, or a variant amino acid sequence thereof.

A number of other methods may be used for generating T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used in the generation of CTL. Plebanski et al. (Plebanski et al., 1995) made use of autologous peripheral blood lymphocytes (PLBs) in the preparation of T cells. Furthermore, the production of autologous T cells by pulsing dendritic cells with peptide or polypeptide, or via infection with recombinant virus is possible. Also, B cells can be used in the production of autologous T cells. In addition, macrophages pulsed with peptide or polypeptide, or infected with recombinant virus, may be used in the preparation of autologous T cells. S. Walter et al. (Walter et al., 2003) describe the in vitro priming of T cells by using artificial antigen presenting cells (aAPCs), which is also a suitable way for generating T cells against the peptide of choice. In the present invention, aAPCs were generated by the coupling of preformed MHC:peptide complexes to the surface of polystyrene particles (microbeads) by biotin:streptavidin biochemistry. This system permits the exact control of the MHC density on aAPCs, which allows to selectively elicit high- or low-avidity antigen-specific T cell responses with high efficiency from blood samples. Apart from MHC:peptide complexes, aAPCs should carry other proteins with co-stimulatory activity like anti-CD28 antibodies coupled to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, e. g. cytokines, like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and a method is described in detail in WO 97/26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells. In addition plant viruses may be used (see, for example, Porta et al. (Porta et al., 1994) which describes the development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides.

The activated T cells that are directed against the peptides of the invention are useful in therapy. Thus, a further aspect of the invention provides activated T cells obtainable by the foregoing methods of the invention.

Activated T cells, which are produced by the above method, will selectively recognize a cell that aberrantly expresses a polypeptide that comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 385.

Preferably, the T cell recognizes the cell by interacting through its TCR with the HLA/peptide-complex (for example, binding). The T cells are useful in a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention wherein the patient is administered an effective number of the activated T cells. The T cells that are administered to the patient may be derived from the patient and activated as described above (i.e. they are autologous T cells). Alternatively, the T cells are not from the patient but are from another individual. Of course, it is preferred if the individual is a healthy individual. By “healthy individual” the inventors mean that the individual is generally in good health, preferably has a competent immune system and, more preferably, is not suffering from any disease that can be readily tested for, and detected.

In vivo, the target cells for the CD8-positive T cells according to the present invention can be cells of the tumor (which sometimes express MHC class II) and/or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredients of a therapeutic composition. Thus, the invention also provides a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, the method comprising administering to the patient an effective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptide is over-expressed compared to levels of expression in normal tissues or that the gene is silent in the tissue from which the tumor is derived but in the tumor it is expressed. By “over-expressed” the inventors mean that the polypeptide is present at a level at least 1.2-fold of that present in normal tissue; preferably at least 2-fold, and more preferably at least 5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. those described above.

Protocols for this so-called adoptive transfer of T cells are well known in the art. Reviews can be found in: Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptides complexed with MHC to generate a T-cell receptor whose nucleic acid is cloned and is introduced into a host cell, preferably a T cell. This engineered T cell can then be transferred to a patient for therapy of cancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T-cell receptor or the nucleic acid encoding it, is useful for the treatment of disorders, characterized by cells escaping an immune response. Therefore, any molecule of the present invention may be used as medicament or in the manufacture of a medicament. The molecule may be used by itself or combined with other molecule(s) of the invention or (a) known molecule(s).

The present invention is further directed at a kit comprising:

-   (a) a container containing a pharmaceutical composition as described     above, in solution or in lyophilized form; -   (b) optionally a second container containing a diluent or     reconstituting solution for the lyophilized formulation; and -   (c) optionally, instructions for (i) use of the solution or (ii)     reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. The container is preferably a bottle, a vial, a syringe or test tube; and it may be a multi-use container. The pharmaceutical composition is preferably lyophilized.

Kits of the present invention preferably comprise a lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and/or use. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. Preferably the kit and/or container contain/s instructions on or associated with the container that indicates directions for reconstitution and/or use. For example, the label may indicate that the lyophilized formulation is to be reconstituted to peptide concentrations as described above. The label may further indicate that the formulation is useful or intended for subcutaneous administration.

The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).

Upon mixing of the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3 mg/mL/peptide (=1500 μg). The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Kits of the present invention may have a single container that contains the formulation of the pharmaceutical compositions according to the present invention with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component.

Preferably, kits of the invention include a formulation of the invention packaged for use in combination with the co-administration of a second compound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenesis agent or inhibitor, an apoptosis-inducing agent or a chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. Usually, when there is more than one component, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, a therapeutic kit will contain an apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the agents of the invention that are components of the present kit.

The present formulation is one that is suitable for administration of the peptides by any acceptable route such as oral (enteral), nasal, ophthal, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, the administration is s.c., and most preferably i.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from CLL, the medicament of the invention is preferably used to treat CLL.

The present invention further relates to a method for producing a personalized pharmaceutical for an individual patient comprising manufacturing a pharmaceutical composition comprising at least one peptide selected from a warehouse of pre-screened TUMAPs, wherein the at least one peptide used in the pharmaceutical composition is selected for suitability in the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method could also be adapted to produce T cell clones for down-stream applications, such as TCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailored therapies for one individual patient that will only be used for therapy in such individual patient, including actively personalized cancer vaccines and adoptive cellular therapies using autologous patient tissue.

As used herein, the term “warehouse” shall refer to a group or set of peptides that have been pre-screened for immunogenicity and/or over-presentation in a particular tumor type. The term “warehouse” is not intended to imply that the particular peptides included in the vaccine have been pre-manufactured and stored in a physical facility, although that possibility is contemplated. It is expressly contemplated that the peptides may be manufactured de novo for each individualized vaccine produced, or may be pre-manufactured and stored. The warehouse (e.g. in the form of a database) is composed of tumor-associated peptides which were highly overexpressed in the tumor tissue of CLL patients with various HLA-A HLA-B and HLA-C alleles. It may contain MHC class I and MHC class II peptides or elongated MHC class I peptides. In addition to the tumor associated peptides collected from several CLL tissues, the warehouse may contain HLA-A*02 and HLA-A*24 marker peptides. These peptides allow comparison of the magnitude of T-cell immunity induced by TUMAPS in a quantitative manner and hence allow important conclusion to be drawn on the capacity of the vaccine to elicit anti-tumor responses. Secondly, they function as important positive control peptides derived from a “non-self” antigen in the case that any vaccine-induced T-cell responses to TUMAPs derived from “self” antigens in a patient are not observed. And thirdly, it may allow conclusions to be drawn, regarding the status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integrated functional genomics approach combining gene expression analysis, mass spectrometry, and T-cell immunology (XPresident®)). The approach assures that only TUMAPs truly present on a high percentage of tumors but not or only minimally expressed on normal tissue, are chosen for further analysis. For initial peptide selection, CLL samples from patients and blood from healthy donors were analyzed in a stepwise approach:

-   1. HLA ligands from the malignant material were identified by mass     spectrometry -   2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis     was used to identify genes over-expressed in the malignant tissue     (CLL) compared with a range of normal organs and tissues -   3. Identified HLA ligands were compared to gene expression data.     Peptides over-presented or selectively presented on tumor tissue,     preferably encoded by selectively expressed or over-expressed genes     as detected in step 2 were considered suitable TUMAP candidates for     a multi-peptide vaccine. -   4. Literature research was performed in order to identify additional     evidence supporting the relevance of the identified peptides as     TUMAPs -   5. The relevance of over-expression at the mRNA level was confirmed     by redetection of selected TUMAPs from step 3 on tumor tissue and     lack of (or infrequent) detection on healthy tissues. -   6. In order to assess, whether an induction of in vivo T-cell     responses by the selected peptides may be feasible, in vitro     immunogenicity assays were performed using human T cells from     healthy donors as well as from CLL patients.

In an aspect, the peptides are pre-screened for immunogenicity before being included in the warehouse. By way of example, and not limitation, the immunogenicity of the peptides included in the warehouse is determined by a method comprising in vitro T-cell priming through repeated stimulations of CD8+ T cells from healthy donors with artificial antigen presenting cells loaded with peptide/MHC complexes and anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rare expression profile. In contrast to multi-peptide cocktails with a fixed composition as currently developed, the warehouse allows a significantly higher matching of the actual expression of antigens in the tumor with the vaccine. Selected single or combinations of several “off-the-shelf” peptides will be used for each patient in a multitarget approach. In theory, an approach based on selection of e.g. 5 different antigenic peptides from a library of 50 would already lead to approximately 17 million possible drug product (DP) compositions.

In one aspect, the peptides are selected for inclusion in the vaccine based on their suitability for the individual patient based on the method according to the present invention as described herein, or as below.

The HLA phenotype, transcriptomic and peptidomic data is gathered from the patient's tumor material, and blood samples to identify the most suitable peptides for each patient containing “warehouse” and patient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen, which are selectively or over-expressed in the patients' tumor and, where possible, show strong in vitro immunogenicity if tested with the patients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by a method comprising: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the individual patient; (b) comparing the peptides identified in (a) with a warehouse (database) of peptides as described above; and (c) selecting at least one peptide from the warehouse (database) that correlates with a tumor-associated peptide identified in the patient. For example, the TUMAPs presented by the tumor sample are identified by: (a1) comparing expression data from the tumor sample to expression data from a sample of normal tissue corresponding to the tissue type of the tumor sample to identify proteins that are over-expressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and/or class II molecules in the tumor sample to identify MHC ligands derived from proteins over-expressed or aberrantly expressed by the tumor. Preferably, the sequences of MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample, and sequencing the eluted ligands. Preferably, the tumor sample and the normal tissue are obtained from the same patient.

In addition to, or as an alternative to, selecting peptides using a warehousing (database) model, TUMAPs may be identified in the patient de novo, and then included in the vaccine. As one example, candidate TUMAPs may be identified in the patient by (a1) comparing expression data from the tumor sample to expression data from a sample of normal tissue corresponding to the tissue type of the tumor sample to identify proteins that are over-expressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and/or class II molecules in the tumor sample to identify MHC ligands derived from proteins over-expressed or aberrantly expressed by the tumor. As another example, proteins may be identified containing mutations that are unique to the tumor sample relative to normal corresponding tissue from the individual patient, and TUMAPs can be identified that specifically target the mutation. For example, the genome of the tumor and of corresponding normal tissue can be sequenced by whole genome sequencing: For discovery of non-synonymous mutations in the protein-coding regions of genes, genomic DNA and RNA are extracted from tumor tissues and normal non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs). The applied NGS approach is confined to the re-sequencing of protein coding regions (exome re-sequencing). For this purpose, exonic DNA from human samples is captured using vendor-supplied target enrichment kits, followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally, tumor mRNA is sequenced for direct quantification of gene expression and validation that mutated genes are expressed in the patients' tumors. The resultant millions of sequence reads are processed through software algorithms. The output list contains mutations and gene expression. Tumor-specific somatic mutations are determined by comparison with the PBMC-derived germline variations and prioritized. The de novo identified peptides can then be tested for immunogenicity as described above for the warehouse, and candidate TUMAPs possessing suitable immunogenicity are selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the individual patient by the method as described above; (b) comparing the peptides identified in a) with a warehouse of peptides that have been prescreened for immunogenicity and overpresentation in tumors as compared to corresponding normal tissue; (c) selecting at least one peptide from the warehouse that correlates with a tumor-associated peptide identified in the patient; and (d) optionally, selecting at least one peptide identified de novo in (a) confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the individual patient; and (b) selecting at least one peptide identified de novo in (a) and confirming its immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected, the vaccine is produced. The vaccine preferably is a liquid formulation consisting of the individual peptides dissolved in between 20-40% DMSO, preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. The concentration of the single peptide solutions has to be chosen depending on the number of peptides to be included into the product. The single peptide-DMSO solutions are mixed in equal parts to achieve a solution containing all peptides to be included in the product with a concentration of ˜2.5 mg/ml per peptide. The mixed solution is then diluted 1:3 with water for injection to achieve a concentration of 0.826 mg/ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. until use. One vial contains 700 μL solution, containing 0.578 mg of each peptide. Of this, 500 μL (approx. 400 μg per peptide) will be applied for intradermal injection.

In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostics. Since the peptides were generated from CLL cells and since it was determined that these peptides are not or at lower levels present in normal tissues, these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples can assist a pathologist in diagnosis of cancer. Detection of certain peptides by means of antibodies, mass spectrometry or other methods known in the art can tell the pathologist that the tissue sample is malignant or inflamed or generally diseased, or can be used as a biomarker for CLL. Presence of groups of peptides can enable classification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable the decision about the benefit of therapies involving the immune system, especially if T-lymphocytes are known or expected to be involved in the mechanism of action. Loss of MHC expression is a well described mechanism by which infected of malignant cells escape immuno-surveillance. Thus, presence of peptides shows that this mechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyze lymphocyte responses against those peptides such as T cell responses or antibody responses against the peptide or the peptide complexed to MHC molecules. These lymphocyte responses can be used as prognostic markers for decision on further therapy steps. These responses can also be used as surrogate response markers in immunotherapy approaches aiming to induce lymphocyte responses by different means, e.g. vaccination of protein, nucleic acids, autologous materials, adoptive transfer of lymphocytes. In gene therapy settings, lymphocyte responses against peptides can be considered in the assessment of side effects. Monitoring of lymphocyte responses might also be a valuable tool for follow-up examinations of transplantation therapies, e.g. for the detection of graft versus host and host versus graft diseases.

The present invention will now be described in the following examples which describe preferred embodiments thereof, and with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

Figures

FIG. 1A to V show the overpresentation of various peptides in normal tissues (white bars) and CLL (black bars). FIG. 1A) Gene: IGHM, Peptide: ALHRPDVYL (SEQ ID NO.: 2). Tissues from left to right: 2 adipose tissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6 esophagi, 17 chronic lymphocytic leukemia samples. The peptide has additionally been detected on 2/21 non-Hodgkin lymphoma samples. FIG. 1B) Gene: IGHM, Peptide: VIAELPPKV (SEQ ID NO.: 3). Tissues from left to right: 2 adipose tissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6 esophagi, 17 chronic lymphocytic leukemia samples. The peptide has additionally been detected on 5/21 non-Hodgkin lymphoma samples and 1/90 lung cancers. FIG. 1C), Gene: IGHM, Peptide: VIAELPPKVSV (SEQ ID No.: 4), Tissues from left to right: 2 adipose tissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6 esophagi, 17 chronic lymphocytic leukemia samples. FIG. 1D), Gene: NUP210, Peptide: RLYEITIEV (SEQ ID No.: 121). Samples from left to right: 1 PBMC culture, 1 benign prostate, 8 normal tissues (3 lungs, 4 spleens, 1 trachea), 49 cancer tissues (2 brain cancers, 2 breast cancers, 1 colon cancer, 13 leukocytic leukemia cancers, 13 lung cancers, 8 lymph node cancers, 1 myeloid cells cancer, 4 ovarian cancers, 2 skin cancers, 1 urinary bladder cancer, 2 uterus cancers). Discrepancies regarding the list of tumor types between FIG. 1D and table 4 may be due to the more stringent selection criteria applied in table 4 (for details please refer to table 4). The normal tissue panel and the cancer cell lines and xenografts tested were the same as in FIG. 1A-C. FIG. 1E), Gene: COBRA1, Peptide: ALLRLLPGL (SEQ ID No.: 14). Samples from left to right: 1 cell line, 11 cancer tissues (1 breast cancer, 1 colon cancer, 1 head and neck cancer, 2 leukocytic leukemia cancers, 1 liver cancer, 1 lung cancer, 2 lymph node cancers, 1 myeloid cell cancer, 1 skin cancer). FIG. 1F), Gene: KIAA0226L, Peptide: GIIDGSPRL (SEQ ID No.: 62). Samples from left to right: 2 normal tissues (1 lymph node, 1 spleen), 18 cancer tissues (2 colon cancers, 9 leukocytic leukemia cancers, 6 lymph node cancers, 1 rectum cancer). FIG. 1G), Gene: DDX3X, Peptide: GLDQQFAGLDL (SEQ ID No.: 68). Samples from left to right: 1 cell line, 2 primary cultures, 21 cancer tissues (1 bile duct cancer, 1 brain cancer, 1 breast cancer, 1 head and neck cancer, 2 kidney cancers, 3 leukocytic leukemia cancers, 2 liver cancers, 2 lung cancers, 2 lymph node cancers, 3 ovarian cancers, 1 rectum cancer, 1 skin cancer, 1 uterus cancer). FIG. 1H), Gene: LIG1, Peptide: KTLDVDATYEI (SEQ ID No.: 112). Samples from left to right: 2 cell lines, 1 primary culture, 13 cancer tissues (1 esophageal cancer, 2 leukocytic leukemia cancers, 1 liver cancer, 3 lung cancers, 2 lymph node cancers, 2 ovarian cancers, 1 skin cancer, 1 uterus cancer). FIG. 1I), Gene: SMCHD1, Peptide: SIIEGPIIKL (SEQ ID No.: 146). Samples from left to right: 14 cancer tissues (1 head and neck cancer, 3 leukocytic leukemia cancers, 6 lung cancers, 1 lymph node cancer, 1 ovarian cancer, 2 urinary bladder cancers). FIG. 1J), Gene: MMS22L, Peptide: SILETVATL (SEQ ID No.: 147). Samples from left to right: 1 primary culture, 11 cancer tissues (1 colon cancer, 4 leukocytic leukemia cancers, 1 lung cancer, 3 lymph node cancers, 1 myeloid cell cancer, 1 skin cancer). FIG. 1K), Gene: FCRL3, Peptide: SLLAELHVL (SEQ ID No.: 153). Samples from left to right: 1 cell line, 9 normal tissues (1 lymph node, 1 rectum, 7 spleens), 27 cancer tissues (1 breast cancer, 1 kidney cancer, 10 leukocytic leukemia cancers, 14 lymph node cancers, 1 skin cancer). FIG. 1L), Gene: FAM126A, Peptide: SLQEEKLIYV (SEQ ID No.: 159). Samples from left to right: 2 cell lines, 23 cancer tissues (5 brain cancers, 1 breast cancer, 1 esophageal cancer, 3 head and neck cancers, 2 leukocytic leukemia cancers, 5 lung cancers, 3 lymph node cancers, 1 skin cancer, 2 urinary bladder cancers). FIG. 1M), Gene: HNRNPC, Peptide: SVHKGFAFV (SEQ ID No.: 164). Samples from left to right: 10 cell lines, 1 primary culture, 1 normal tissue (1 adrenal gland), 36 cancer tissues (12 brain cancers, 1 colon cancer, 2 head and neck cancers, 2 kidney cancers, 3 leukocytic leukemia cancers, 1 liver cancer, 5 lung cancers, 1 myeloid cell cancer, 1 ovarian cancer, 1 rectum cancer, 1 skin cancer, 2 stomach cancers, 2 urinary bladder cancers, 2 uterus cancers). FIG. 1N), Gene: RASGRF1, Peptide: TLDTSKLYV (SEQ ID No.: 167). Samples from left to right: 13 cancer tissues (1 brain cancer, 8 leukocytic leukemia cancers, 4 lymph node cancers). FIG. 1O), Gene: RASGRF1, Peptide: YLLDQSFVM (SEQ ID No.: 168). Samples from left to right: 16 cancer tissues (10 leukocytic leukemia cancers, 6 lymph node cancers). FIG. 1P), Gene: RNF213, Peptide: YIQEYLTLL (SEQ ID No.: 192). Samples from left to right: 2 cell lines, 18 cancer tissues (1 colon cancer, 1 head and neck cancer, 1 kidney cancer, 4 leukocytic leukemia cancers, 5 lung cancers, 3 lymph node cancers, 1 myeloid cell cancer, 1 stomach cancer, 1 urinary bladder cancer). FIG. 1Q), Gene: DCK, Peptide: YLQEVPILTL (SEQ ID No.: 197). Samples from left to right: 11 cancer tissues (5 leukocytic leukemia cancers, 4 lymph node cancers, 1 skin cancer, 1 uterus cancer). FIG. 1R), Gene: EIF3H, Peptide: YMFEEVPIVI (SEQ ID No.: 200). Samples from left to right: 4 cell lines, 2 primary cultures, 34 cancer tissues (1 bile duct cancer, 2 breast cancers, 1 colon cancer, 1 esophageal cancer, 1 gallbladder cancer, 5 head and neck cancers, 2 leukocytic leukemia cancers, 2 liver cancers, 9 lung cancers, 4 lymph node cancers, 2 ovarian cancers, 1 rectum cancer, 2 skin cancers, 1 urinary bladder cancer). FIG. 1S), Gene: UBLCP1, Peptide: LIDVKPLGV (SEQ ID No.: 211). Samples from left to right: 3 cell lines, 23 cancer tissues (5 brain cancers, 1 breast cancer, 1 esophageal cancer, 1 head and neck cancer, 1 kidney cancer, 3 leukocytic leukemia cancers, 2 liver cancers, 2 lung cancers, 4 lymph node cancers, 1 ovarian cancer, 1 prostate cancer, 1 skin cancer). FIG. 1T), Gene: TMCO6, Peptide: QLLPVSNVVSV (SEQ ID No.: 256). Samples from left to right: 2 primary cultures, 13 cancer tissues (2 head and neck cancers, 3 leukocytic leukemia cancers, 1 liver cancer, 2 lung cancers, 4 lymph node cancers, 1 ovarian cancer). FIG. 1U), Gene: MYO1G, Peptide: LLYNSTDPTL (SEQ ID No.: 312). Samples from left to right: 1 cell line, 2 primary cultures, 14 cancer tissues (1 colon cancer, 4 leukocytic leukemia cancers, 2 lung cancers, 5 lymph node cancers, 1 ovarian cancer, 1 urinary bladder cancer). FIG. 1V), Gene: PIM1, Peptide: VLLPQETAEIHL (SEQ ID No.: 328). Samples from left to right: 3 primary cultures, 27 cancer tissues (1 breast cancer, 1 colon cancer, 5 leukocytic leukemia cancers, 5 lung cancers, 7 lymph node cancers, 2 ovarian cancers, 2 skin cancers, 1 stomach cancer, 3 urinary bladder cancers).

FIGS. 2A to D show exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in CLL in a panel of normal tissues (white bars) and 17 CLL samples (black bars). Tissues from left to right: 6 arteries, 1 blood cells, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 6 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 5 pancreases, 2 pituitary glands, 1 rectum, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1 thymus, 1 uterus, 10 chronic lymphocytic leukemia samples. FIG. 2A) Gene symbol: FCER2; FIG. 2B) Gene symbol: KIAA0226L; FIG. 2C) Gene symbol: PAX5; FIG. 2D) Gene symbol: CLEC17A.

FIG. 3 shows exemplary immunogenicity data: flow cytometry results after peptide-specific multimer staining. CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex with SeqID No 1 peptide (C, left panel), SeqID No 5 peptide (D left panel), SeqID No 32 peptide (E, left panel) and SeqID No 220 peptide (F, left panel), respectively. After three cycles of stimulation, the detection of peptide-reactive cells was performed by 2D multimer staining with A*02/SeqID No 1 (C), A*02/SeqID No 5 (D), A*02/SeqID No 32 (E) or A*02/SeqID No 220 (F). Right panels (C, D, E and F) show control staining of cells stimulated with irrelevant A*02/peptide complexes. Viable singlet cells were gated for CD8+ lymphocytes. Boolean gates helped excluding false-positive events detected with multimers specific for different peptides. Frequencies of specific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presented on the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: ProteoGenex Inc. (Culver City, Calif., USA); University Hospital Bonn (Bonn, Germany); University Hospital Tübingen (Tübingen, Germany).

Normal tissues were obtained from Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); BioServe (Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA); Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva (Geneva, Switzerland); University Hospital Heidelberg (Heidelberg, Germany); University Hospital Munich (Munich, Germany); University Hospital Tübingen (Tubingen, Germany).

Written informed consents of all patients had been given before surgery or autopsy. Tissues were shock-frozen immediately after excision and stored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained by immune precipitation from solid tissues according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibody W6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPLC system, Waters) and the eluting peptides were analyzed in LTQ-velos and fusion hybrid mass spectrometers (ThermoElectron) equipped with an ESI source. Peptide pools were loaded directly onto the analytical fused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) applying a flow rate of 400 nL per minute. Subsequently, the peptides were separated using a two-step 180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of

Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometers were operated in the data-dependent mode using a TOP5 strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the orbitrap (R=30 000), which was followed by MS/MS scans also in the orbitrap (R=7500) on the 5 most abundant precursor ions with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST and additional manual control. The identified peptide sequence was assured by comparison of the generated natural peptide fragmentation pattern with the fragmentation pattern of a synthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion counting i.e. by extraction and analysis of LC-MS features (Mueller et al., 2007). The method assumes that the peptide's LC-MS signal area correlates with its abundance in the sample. Extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with the sequence identification results to combine quantitative data of different samples and tissues to peptide presentation profiles. The quantitative data were normalized in a two-tier fashion according to central tendency to account for variation within technical and biological replicates. Thus each identified peptide can be associated with quantitative data allowing relative quantification between samples and tissues. In addition, all quantitative data acquired for peptide candidates was inspected manually to assure data consistency and to verify the accuracy of the automated analysis. For each peptide, a presentation profile was calculated showing the mean sample presentation as well as replicate variations. The profiles juxtapose CLL samples to a baseline of normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown in FIG. 1. Presentation scores for exemplary peptides are shown in Table 8.

TABLE 8 Presentation scores. The table lists peptides that are very highly over-presented on tumors compared to a panel of normal tissues (+++), highly over-presented on tumors compared to a panel of normal tissues (++) or over-presented on tumors compared to a panel of normal tissues (+). The panel of normal tissues considered relevant for comparison with tumors consisted of: adipose tissue, adrenal gland, blood cells, blood vessel, bone marrow, brain, breast, esophagus, eye, gall- bladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter, urinary bladder. SEQ ID Sequence No. Presentation Peptide 1 AIPPSFASIFL +++ 2 ALHRPDVYL +++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 6 ALDTLEDDMTI +++ 7 ALLERTGYTL +++ 8 ALAASALPALV +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 11 ALFTFJPLTV +++ 12 ALGEDEITL +++ 13 VVDGMPPGV ++ 14 ALLRLLPGL + 15 ALPEVSVEA + 16 ALPGGAAVAAV ++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV + 20 ALVDPGPDFVV + 21 ALWAGLLTL +++ 22 ALWDPVIEL +++ 23 ALYLTEVFL +++ 24 AMAGDVVYA +++ 25 ATYSGLESQSV + 27 AVLGLVWLL ++ 28 AVLHLLLSV +++ 29 AVLQAVTAV +++ 30 ELLEGSEIYL + 31 ELMHGVAGV ++ 32 FIDKFTPPV +++ 33 FIINSSNIFL +++ 35 FILPSSLYL +++ 36 FILTHVDQL +++ 37 FIMEGGAMVL +++ 39 FLDALLTDV +++ 40 FLDEDDMSL + 41 FLDPSLDPLL +++ 43 FLLGPEALSFA +++ 45 FLPELPADLEA ++ 47 FLSDQPEPYL ++ 48 FLSPQQPPLL +++ 49 FLTDLFAQL + 50 FLFEPVVKAFL +++ 51 FLVEAPHDWDL ++ 52 FLVETGFLHV +++ 53 FLWQHVELVL +++ 54 FLYPFPLALF +++ 56 FVFEAPYTL +++ 57 GLSEISLRL +++ 58 YIQQGIFSV ++ 59 FVFGDENGTVSL +++ 60 FVLDHEDGLNL ++ 61 FVYFIVREV +++ 62 GIIDGSPRL + 63 SLAHVAGCEL +++ 64 KLLESVASA ++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 68 GLDQQFAGLDL +++ 69 GLHQREIFL +++ 70 FVPDTPVGV ++ 71 GLKHDIARV +++ 72 GLLDAGKMYV +++ 73 GLLEVISALQL +++ 74 GLLRASFLL +++ 76 GLLRIIPYL ++ 77 GLLRLTWFL ++ 78 GLPSFLTEV +++ 79 GLQAKIQEA +++ 80 VLIEDELEEL +++ 81 WLVGQEFEL +++ 82 GLQSGVDIGV + 83 GQGEVLVYV + 86 HLYPGAVTI +++ 88 ILDEIGADVQA +++ 89 ILDFGTFQL +++ 90 VIADLGLIRV +++ 92 ILEPLNPLL +++ 93 ILFNTQINI +++ 94 ILFPLRFTL +++ 95 ILGYMAHEHKV +++ 96 ILIDKTSFV + 97 LLFATQITL + 98 SLIKYFLFV +++ 99 ILIFHSVAL +++ 100 ILNNEVFAI + 101 ILVVIEPLL +++ 102 IQDRAVPSL +++ 103 KLGGTPAPA ++ 104 KLILLDTPLFL +++ 105 KLMNDIADI +++ 106 FMASHLDYL +++ 107 ILYNLYDLL ++ 108 VIYTLIHYI + 109 KLWEGLTELV ++ 112 KTLDVDATYEI +++ 113 KVPAEEVLVAV + 114 LIPEGPPQV +++ 115 LLFDKLYLL +++ 116 LLIGATMQV +++ 117 LLILENILL ++ 118 RLLILENILL +++ 119 VLPAEFFEV +++ 121 RLYEITIEV ++ 122 LLIPVVPGV ++ 123 LLLAEAELLTL +++ 124 LLLEETEKQAV ++ 125 LLLEIGEVGKLFV ++ 126 LLPEGGITAI +++ 127 LLPTAPTTV +++ 128 LLSEEEYHL +++ 129 LLVGTLDVV + 130 LLVLIPVYL +++ 131 LQALEVLKI +++ 132 LVYEAIIMV +++ 133 YLLSGDISEA +++ 134 MLLEHGITLV +++ 135 MTAGFSTIAGSV +++ 137 NLIKTVIKL + 138 NLLDIDAPVTV ++ 139 NLTDVVEKL +++ 140 QIAELPATSV +++ 141 QILSEIVEA ++ 142 QLDEPAPQV +++ 144 QLPPFPREL +++ 145 SALDTITTV +++ 146 SIIEGPIIKL +++ 147 SILETVATL +++ 148 SIVASLITV +++ 149 SLDNGGYYI +++ 153 SLLAELHVL ++ 154 SLLAELHVLTV ++ 155 SLMLEVPAL +++ 157 SLNIRDFTM +++ 160 SLSFLVPSL +++ 162 SMKDDLENV +++ 163 SQLDISEPYKV + 164 SVHKGFAFV +++ 165 TLDDDLDTV +++ 166 TLDPNQVSL +++ 167 TLDTSKLYV +++ 168 YLLDQSFVM +++ 169 TLLLGLTEV +++ 170 TLTFRVETV +++ 171 TLVPPAALISI +++ 172 TLYDMLASI +++ 174 VLAELPIIVV +++ 175 FTVPRVVAV ++ 176 VLAEQNIIPSA +++ 177 VLDDRELLL +++ 178 VLFFNVQEV +++ 179 VLLGLEMTL +++ 181 VLLSIPFVSV + 182 VLLSVPGPPV +++ 185 VMDDQRDLI + 186 VMDPTKILI +++ 187 VMDTHLVNI +++ 189 VMLEMTPEL +++ 190 VVMGTVPRL +++ 191 YIFDGSDGGV +++ 192 YIQEYLTLL +++ 193 YLDLSNNRL +++ 194 YLDNVLAEL + 195 YLGGFALSV +++ 197 YLQEVPILTL +++ 198 YLTFLPAEV +++ 199 YLVELSSLL +++ 200 YMFEEVPIVI +++ 201 YQLELHGIEL +++ 202 YVDDVFLRV +++ 204 GLLQINDKIAL ++ 205 GLSQANFTL + 206 HMQDVRVLL +++ 208 ILLKTEGINL ++ 209 ILQAELPSL ++ 210 KLLVQDFFL +++ 211 LIDVKPLGV +++ 212 NIIEAINELLV ++ 213 RLLYQLVFL ++ 214 RLQELTEKL + 215 VMQDIVYKL + 217 ALDEPPYLTV + 218 ALGEEWKGYVV + 219 ALLNLLESA ++ 221 ALVSTIIMV + 225 ILQERELLPV + 226 AMNISVPQV + 227 FLAEASVMTQL ++ 228 FLGGLSPGV ++ 229 FLLNLQNCHL ++ 231 FLYIRQLAI + 232 FMHQIIDQV + 234 GLDDAEYAL + 235 GLDDLLLFL + 236 GLLESGRHYL +++ 237 GLQENLDVVV + 238 GLVETELQL + 240 ILARDILEI ++ 241 ILGDILLKV + 242 ILLGIQELL ++ 243 ILPTLEKELFL + 244 ILQALAVHL ++ 245 KIMDYSLLLGV + 248 KTVEPPISQV + 249 LLPTGVFQV + 251 LLYDNVPGA + 254 QLIPKLIFL +++ 255 YLFEEAISM + 258 RLDYITAEI ++ 259 RLLDEQFAVL + 260 SLDDVEGMSV ++ 261 SLVEAQGWLV ++ 263 SQWEDIHVV ++ 264 TILDYINVV +++ 266 TLLDQLDTQL + 267 TLLDWQDSL + 268 TLLQVFHLL ++ 270 TVLPVPPLSV +++ 271 VIRNIVEAA ++ 273 VLGEYSYLL + 275 VLLFIEHSV + 276 VLNDGAPNV + 277 VMILKLPFL + 278 YLDDLLPKL ++ 280 YTLDSLYWSV + 281 NLLDDRGMTAL +++ 282 LLRDGIELV +++ 283 ILQPMDIHV +++ 284 LLSAAEPVPA +++ 286 FLLEDLSQKL +++ 287 FLWEEKFNSL +++ 289 ILEEQPMDMLL +++ 290 LANPHELSL +++ 291 ILLNEDDLVTI +++ 292 AAALIIHHV + 294 ALLDQLHTLL + 297 FLVEPQEDTRL ++ 298 IILPVEVEV +++ 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 302 ILQDLTFVHL + 304 LAIVPVNTL +++ 305 LLFPQIEGIKI + 307 LLLTKPTEA ++ 308 SLYDVSRMYV +++ 309 ILYGTQFVL + 310 LLSTLHLLV + 311 LLVDVEPKV + 312 LLYNSTDPTL +++ 314 LMKDCEAEV ++ 316 MLLEHGITL +++ 317 NLLAHIWAL + 318 NLQVTQPTV +++ 320 TIAPVTVAV + 322 SLASIHVPL +++ 323 SLDLFNCEV +++ 327 VLIKWFPEV +++ 328 VLLPQETAEIHL ++ 329 VLMDGSVKL + 330 VLMWEIYSL ++ 331 VLWELAHLPTL +++ 332 VMIQHVENL +++ 334 YLLEEKIASL +++ 336 YMAVTTQEV +++ 337 YMYEKESEL ++ 338 FLDMTNWNL + 339 GLWGTVVNI + 340 KLLEEICNL ++ 341 LLAELPASVHA ++ 342 SLITPLQAV +++ 343 TLLEALDCI ++ 344 VLAFENPQV ++ 345 YLIEPDVELQRI + 346 VLVQVSPSL +++ 347 YLGPVSPSL + 349 ALATHILSL ++ 350 ALEDRVWEL + 351 ALSEKLARL + 352 ALVFELHYV + 353 ATPMPTPSV ++ 354 FIMDDPAGNSYL + 355 FIWPMLIHI +++ 356 FLHDHQAEL + 357 FLIQEIKTL ++ 358 FLTDYLNDL + 359 FMQDPMEVFV ++ 360 HLIDTNKIQL + 362 ILTELGGFEV + 363 ITTEVVNELYV + 364 KMDWIFHTI + 365 LISPLLLPV ++ 367 NLWSLVAKV ++ 368 QLQPTDALLCV + 371 SLADDSVLERL + 372 SLFGPLPGPGPALV + 374 VLSVITEEL ++ 375 VLWFKPVEL ++ 377 VVDGTCVAV + 378 YILGKFFAL + 379 YLAELVTPIL + 380 YLDRKLLTL + 382 YLLPLLQRL +++ 383 YLLREWVNL +++ 384 YMIGSEVGNYL + 385 YTIPLAIKL +++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its usefulness in immunotherapy, and some peptides are tumor-specific despite their source protein occurring also in normal tissues. Still, mRNA expression profiling adds an additional level of safety in selection of peptide targets for immunotherapies. Especially for therapeutic options with high safety risks, such as affinity-matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and not found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above (see Example 1) after written informed consent had been obtained from each patient. Tumor tissue specimens were snap-frozen immediately after surgery and later homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA); University Hospital Heidelberg (Heidelberg, Germany).

Total RNA from tumor tissues for RNASeq experiments was obtained from: Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn, Germany)

Quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples was performed by next generation sequencing (RNAseq) by CeGaT (Tübingen, Germany). Briefly, sequencing libraries are prepared using the Illumina HiSeq v4 reagent kit according to the provider's protocol (Illumina Inc, San Diego, Calif., USA), which includes RNA fragmentation, cDNA conversion and addition of sequencing adaptors. Libraries derived from multiple samples are mixed equimolarly and sequenced on the Illumina HiSeq 2500 sequencer according to the manufacturer's instructions, generating 50 bp single end reads. Processed reads are mapped to the human genome (GRCh38) using the STAR software. Expression data are provided on transcript level as RPKM (Reads Per Kilobase per Million mapped reads, generated by the software Cufflinks) and on exon level (total reads, generated by the software Bedtools), based on annotations of the ensembl sequence database (Ensembl77). Exon reads are normalized for exon length and alignment size to obtain RPKM values. Exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in CLL are shown in FIG. 3. Expression scores for further exemplary genes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptides from genes that are very highly over-expressed in tumors compared to a panel of normal tissues (+++), highly over-expressed in tumors compared to a panel of normal tissues (++) or over-expressed in tumors compared to a panel of normal tissues (+). The baseline for this score was calculated from measurements of the following relevant normal tissues: adipose tissue, adrenal gland, artery, blood cells, bone marrow, brain, cartilage, colon, esophagus, eye, gall- bladder, heart, kidney, liver, lung, lymph node, pancreas, pituitary, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary bladder, and vein. In case expression data for several samples of the same tissue type were available, the arithmetic mean of all respective samples was used for the calculation. Gene SEQ ID No Sequence Expression 1 AIPPSFASIFL ++ 2 ALHRPDVYL ++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV +++ 21 ALWAGLLTL +++ 24 AMAGDVVYA +++ 31 ELMHGVAGV +++ 46 YIIDSAQAV ++ 62 GIIDGSPRL +++ 63 SLAHVAGCEL +++ 64 KLLESVASA +++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 71 GLKHDIARV +++ 74 GLLRASFLL ++ 75 GLSIFAQDLRL ++ 85 HLMLHTAAL + 86 HLYPGAVTI +++ 91 ILDLNTYNV + 96 ILIDKTSFV +++ 97 LLFATQITL +++ 98 SLIKYFLFV +++ 105 KLMNDIADI ++ 106 FMASHLDYL + 107 ILYNLYDLL ++ 108 VIYTLIHYI + 113 KVPAEEVLVAV ++ 116 LLIGATMQV + 117 LLILENILL ++ 118 RLLILENILL ++ 119 VLPAEFFEV ++ 120 AIDAALTSV ++ 121 RLYEITIEV ++ 127 LLPTAPTTV + 137 NLIKTVIKL + 146 SIIEGPIIKL +++ 149 SLDNGGYYI +++ 150 SLFDQPLSII +++ 153 SLLAELHVL +++ 154 SLLAELHVLTV +++ 156 SLNIGDVQL +++ 163 SQLDISEPYKV +++ 177 VLDDRELLL ++ 189 VMLEMTPEL +++ 193 YLDLSNNRL + 195 YLGGFALSV ++ 205 GLSQANFTL +++ 206 HMQDVRVLL +++ 213 RLLYQLVFL +++ 220 ALPEILFAKV ++ 221 ALVSTIIMV + 223 ALWVSQPPEI ++ 235 GLDDLLLFL + 251 LLYDNVPGA +++ 254 QLIPKLIFL +++ 255 YLFEEAISM +++ 257 RIINGIIISV +++ 262 SLWNAGTSV ++ 275 VLLFIEHSV ++ 284 LLSAAEPVPA +++ 285 GVATAGCVNEV +++ 286 FLLEDLSQKL ++ 294 ALLDQLHTLL + 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 301 AASPIITLV + 307 LLLTKPTEA ++ 308 SLYDVSRMYV + 309 ILYGTQFVL ++ 312 LLYNSTDPTL + 319 QVIPQLQTV ++ 320 TIAPVTVAV ++ 330 VLMWEIYSL ++ 332 VMIQHVENL +++ 337 YMYEKESEL +++ 352 ALVFELHYV +++ 356 FLHDHQAEL + 360 HLIDTNKIQL + 378 YILGKFFAL + 380 YLDRKLLTL ++

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of the TUMAPs of the present invention, the inventors performed investigations using an in vitro T-cell priming assay based on repeated stimulations of CD8+ T cells with artificial antigen presenting cells (aAPCs) loaded with peptide/MHC complexes and anti-CD28 antibody. This way the inventors could show immunogenicity for HLA-A*0201 restricted TUMAPs of the invention, demonstrating that these peptides are T-cell epitopes against which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigen presenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products via positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtained from the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium (TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/ml Streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations and readout was performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung et al., 1987) was chemically biotinylated using Sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidin coated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 523) from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 524), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of 4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in a volume of 200 μl. Stimulations were initiated in 96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washed coated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3 days at 37° C. Half of the medium was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and incubating was continued for 4 days at 37° C. This stimulation cycle was performed for a total of three times. For the pMHC multimer readout using 8 different pMHC molecules per condition, a two-dimensional combinatorial coding approach was used as previously described (Andersen et al., 2012) with minor modifications encompassing coupling to 5 different fluorochromes. Finally, multimeric analyses were performed by staining the cells with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer equipped with appropriate lasers and filters was used. Peptide specific cells were calculated as percentage of total CD8+ cells. Evaluation of multimeric analysis was done using the FlowJo software (Tree Star, Oregon, USA). In vitro priming of specific multimer+ CD8+ lymphocytes was detected by comparing to negative control stimulations. Immunogenicity for a given antigen was detected if at least one evaluable in vitro stimulated well of one healthy donor was found to contain a specific CD8+ T-cell line after in vitro stimulation (i.e. this well contained at least 1% of specific multimer+ among CD8+ T-cells and the percentage of specific multimer+ cells was at least 10× the median of the negative control stimulations).

In Vitro Immunogenicity for CLL Peptides

For tested HLA class I peptides, in vitro immunogenicity could be demonstrated by generation of peptide specific T-cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for two peptides of the invention are shown in FIG. 3 together with corresponding negative controls. Results for two peptides from the invention are summarized in Table 10A, others in Table 10B.

TABLE 10A in vitro immunogenicity of HLA class I peptides of the invention Seq ID Sequence wells 427 LLYDAVHIV ++ 521 YLYGQTTTYL ++ Exemplary results of in vitro immunogenicity experiments conducted by the applicant for the peptides of the invention. <20% = +; 20% - 49% = ++; 50% - 69% = +++; > = 70% = ++++

TABLE 10B In vitro immunogenicity of additional HLA class I peptides of the invention SEQ ID NO: Sequence Wells positive [%] 1 AIPPSFASIFL “+++” 2 ALHRPDVYL “+” 5 ALIFKIASA “+++” 17 ALTKTNLQL “+” 26 AVLLVLPLV “+” 32 FIDKFTPPV “++++” 49 FLTDLFAQL “++” 54 FLYPFPLALF “+” 56 FVFEAPYTL “++” 57 GLSEISLRL “+” 69 GLHQREIFL “+” 81 WLVGQEFEL “+” 86 HLYPGAVTI “+” 96 ILIDKTSFV “+” 100 ILNNEVFAI “++” 101 ILVVIEPLL “+” 102 IQDRAVPSL “+” 112 KTLDVDATYEI “+” 114 LIPEGPPQV “+” 154 SLLAELHVLTV “+” 192 YIQEYLTLL “+” 197 YLQEVPILTL “+” 200 YMFEEVPIVI “+” 201 YQLELHGIEL “+” 202 YVDDVFLRV “++” 220 ALPEILFAKV “++” 240 ILARDILEI “+” 281 NLLDDRGMTAL “+” 282 LLRDGIELV “++” 307 LLLTKPTEA “+” 320 TIAPVTVAV “+” 322 SLASIHVPL “+” 323 SLDLFNCEV “+” 329 VLMDGSVKL “+++” 332 VMIQHVENL “+” 380 YLDRKLLTL “++++” Exemplary results of in vitro immunogenicity experiments conducted by the applicant for HLA-A*02 restricted peptides of the invention. Results of in vitro immunogenicity experiments are indicated. Percentage of positive wells and donors (among evaluable) are summarized as indicated <20% = +; 20% - 49% = ++; 50% - 69% = +++; > = 70% = ++++

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solid phase peptide synthesis using the Fmoc-strategy. Identity and purity of each individual peptide have been determined by mass spectrometry and analytical RP-HPLC. The peptides were obtained as white to off-white lyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the present invention were further tested for their MHC binding capacity (affinity). The individual peptide-MHC complexes were produced by UV-ligand exchange, where a UV-sensitive peptide is cleaved upon UV-irradiation, and exchanged with the peptide of interest as analyzed. Only peptide candidates that can effectively bind and stabilize the peptide-receptive MHC molecules prevent dissociation of the MHC complexes. To determine the yield of the exchange reaction, an ELISA was performed based on the detection of the light chain (β2m) of stabilized MHC complexes. The assay was performed as generally described in Rodenko et al. (Rodenko et al., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/ml streptavidin in PBS at room temperature, washed 4× and blocked for 1 h at 37° C. in 2% BSA containing blocking buffer. Refolded HLA-A*02:01/MLA-001 monomers served as standards, covering the range of 15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction were diluted 100 fold in blocking buffer. Samples were incubated for 1 h at 37° C., washed four times, incubated with 2 ug/ml HRP conjugated anti-β2m for 1 h at 37° C., washed again and detected with TMB solution that is stopped with NH2SO4. Absorption was measured at 450 nm. Candidate peptides that show a high exchange yield (preferably higher than 50%, most preferred higher than 75%) are generally preferred for a generation and production of antibodies or fragments thereof, and/or T cell receptors or fragments thereof, as they show sufficient avidity to the MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50 = +++; ≥75% = ++++ SEQID Sequence Peptide Exchange 1 AIPPSFASIFL “+++” 2 ALHRPDVYL “+++” 3 VIAELPPKV “++” 4 VIAELPPKVSV “+++” 5 ALIFKIASA “+++” 6 ALDTLEDDMTI “+++” 7 ALLERTGYTL “+++” 8 ALAASALPALV “+++” 9 ALCDTLITV “+++” 10 FVYGESVEL “+++” 11 ALFTFJPLTV “+++” 12 ALGEDEITL “+++” 13 VVDGMPPGV “+++” 14 ALLRLLPGL “+++” 15 ALPEVSVEA “+++” 16 ALPGGAAVAAV “+++” 17 ALTKTNLQL “+++” 18 LLGEFSIKM “+++” 19 QVMEKLAAV “+++” 20 ALVDPGPDFVV “+++” 21 ALWAGLLTL “+++” 22 ALWDPVIEL “+++” 23 ALYLTEVFL “++++” 24 AMAGDVVYA “+++” 25 ATYSGLESQSV “++” 26 AVLLVLPLV “+++” 27 AVLGLVWLL “+++” 28 AVLHLLLSV “++” 29 AVLQAVTAV “+++” 30 ELLEGSEIYL “+++” 31 ELMHGVAGV “+++” 32 FIDKFTPPV “++++” 34 YLPYIFPNI “+++” 35 FILPSSLYL “+++” 36 FILTHVDQL “++++” 37 FIMEGGAMVL “++++” 38 FIMEGGAMV “+++” 39 FLDALLTDV “+++” 40 FLDEDDMSL “++” 41 FLDPSLDPLL “+++” 42 FLEEGGVVTV “+++” 43 FLLGPEALSFA “++++” 44 FLLSINDFL “+++” 45 FLPELPADLEA “+++” 46 YIIDSAQAV “+++” 47 FLSDQPEPYL “++” 48 FLSPQQPPLL “+++” 49 FLTDLFAQL “++++” 50 FLFEPVVKAFL “+++” 51 FLVEAPHDWDL “++++” 52 FLVETGFLHV “++++” 53 FLWQHVELVL “+++” 54 FLYPFPLALF “+++” 55 FMEPTLLML “+++” 56 FVFEAPYTL “+++” 57 GLSEISLRL “++++” 58 YIQQGIFSV “+++” 59 FVFGDENGTVSL “+++” 60 FVLDHEDGLNL “+++” 61 FVYFIVREV “+++” 62 GIIDGSPRL “+++” 63 SLAHVAGCEL “+++” 64 KLLESVASA “+++” 65 GLDDMKANL “++” 66 SLAGGLDDMKA “+++” 67 GLDDVTVEV “++” 68 GLDQQFAGLDL “+” 69 GLHQREIFL “+++” 70 FVPDTPVGV “+++” 71 GLKHDIARV “+++” 72 GLLDAGKMYV “+++” 73 GLLEVISALQL “+++” 74 GLLRASFLL “++” 75 GLSIFAQDLRL “++” 76 GLLRIIPYL “+++” 78 GLPSFLTEV “+++” 79 GLQAKIQEA “+++” 80 VLIEDELEEL “++” 81 WLVGQEFEL “+++” 82 GLQSGVDIGV “+++” 83 GQGEVLVYV “+++” 84 GVMDVNTAL “++” 85 HLMLHTAAL “++++” 86 HLYPGAVTI “+++” 87 HQIEAVDGEEL “++” 88 ILDEIGADVQA “+++” 89 ILDFGTFQL “+++” 90 VIADLGLIRV “+++” 91 ILDLNTYNV “+++” 92 ILEPLNPLL “+++” 93 ILFNTQINI “+++” 94 ILFPLRFTL “+++” 95 ILGYMAHEHKV “++” 96 ILIDKTSFV “+++” 97 LLFATQITL “++” 98 SLIKYFLFV “+” 99 ILIFHSVAL “+++” 100 ILNNEVFAI “+++” 101 ILVVIEPLL “++++” 102 IQDRAVPSL “++” 103 KLGGTPAPA “++” 104 KLILLDTPLFL “+++” 105 KLMNDIADI “++” 106 FMASHLDYL “+++” 107 ILYNLYDLL “+++” 108 VIYTLIHYI “+++” 109 KLWEGLTELV “+++” 110 LLFDHLEPMEL “+++” 111 KLWNVAAPLYL “+++” 112 KTLDVDATYEI “+++” 113 KVPAEEVLVAV “+++” 114 LIPEGPPQV “+++” 115 LLFDKLYLL “+++” 116 LLIGATMQV “+++” 117 LLILENILL “+++” 118 RLLILENILL “+++” 119 VLPAEFFEV “+++” 120 AIDAALTSV “+++” 121 RLYEITIEV “++++” 122 LLIPVVPGV “+++” 123 LLLAEAELLTL “++” 124 LLLEETEKQAV “+++” 125 LLLEIGEVGKLFV “+++” 126 LLPEGGITAI “+++” 127 LLPTAPTTV “+++” 128 LLSEEEYHL “+++” 130 LLVLIPVYL “+” 131 LQALEVLKI “+” 132 LVYEAIIMV “+++” 133 YLLSGDISEA “+++” 134 MLLEHGITLV “++++” 135 MTAGFSTIAGSV “+” 136 NLDKLWTLV “++” 137 NLIKTVIKL “+++” 138 NLLDIDAPVTV “+++” 139 NLTDVVEKL “+++” 140 QIAELPATSV “+++” 141 QILSEIVEA “++” 142 QLDEPAPQV “++” 143 QLLDTYFTL “+++” 145 SALDTITTV “+++” 146 SIIEGPIIKL “+++” 147 SILETVATL “+++” 148 SIVASLITV “+++” 149 SLDNGGYYI “+++” 150 SLFDQPLSII “+++” 151 SLFDSAYGA “+++” 152 SLIRILQTI “+++” 153 SLLAELHVL “+++” 154 SLLAELHVLTV “++++” 155 SLMLEVPAL “+++” 156 SLNIGDVQL “+++” 157 SLNIRDFTM “+++” 158 SLPEAPLDV “+++” 159 SLQEEKLIYV “+++” 160 SLSFLVPSL “+++” 161 SMDDGMINV “+++” 162 SMKDDLENV “++” 163 SQLDISEPYKV “+++” 164 SVHKGFAFV “+++” 165 TLDDDLDTV “++” 166 TLDPNQVSL “++” 167 TLDTSKLYV “+++” 168 YLLDQSFVM “++++” 169 TLLLGLTEV “+++” 170 TLTFRVETV “+++” 171 TLVPPAALISI “+++” 172 TLYDMLASI “+++” 173 TVIENIHTI “++” 174 VLAELPIIVV “+++” 175 FTVPRVVAV “+++” 176 VLAEQNIIPSA “+++” 177 VLDDRELLL “+++” 178 VLFFNVQEV “+++” 179 VLLGLEMTL “+++” 180 LLKDGPEIGL “++” 181 VLLSIPFVSV “+++” 182 VLLSVPGPPV “+++” 183 VLMPTVYQQGV “+++” 184 VLSHNLYTV “+++” 185 VMDDQRDLI “++” 186 VMDPTKILI “++++” 187 VMDTHLVNI “++++” 188 VMGDIPAAV “++” 189 VMLEMTPEL “+++” 190 VVMGTVPRL “+++” 191 YIFDGSDGGV “+++” 192 YIQEYLTLL “+++” 193 YLDLSNNRL “+++” 194 YLDNVLAEL “++++” 195 YLGGFALSV “++++” 196 YLLLQTYVL “+” 197 YLQEVPILTL “++++” 198 YLTFLPAEV “+++” 199 YLVELSSLL “++++” 200 YMFEEVPIVI “++++” 201 YQLELHGIEL “++++” 202 YVDDVFLRV “++++” 203 ALLSSQLAL “+++” 204 GLLQINDKIAL “+++” 205 GLSQANFTL “+++” 206 HMQDVRVLL “+++” 207 IIADLDTTIMFA “+++” 208 ILLKTEGINL “+++” 209 ILQAELPSL “+++” 210 KLLVQDFFL “+++” 211 LIDVKPLGV “+++” 214 RLQELTEKL “+++” 215 VMQDIVYKL “+++” 216 WLAGDVPAA “+++” 217 ALDEPPYLTV “+++” 218 ALGEEWKGYVV “++” 219 ALLNLLESA “+++” 220 ALPEILFAKV “+++” 221 ALVSTIIMV “+” 222 ALWELSLKI “+++” 223 ALWVSQPPEI “+++” 224 AMEALVVEV “+++” 225 ILQERELLPV “+++” 226 AMNISVPQV “+++” 227 FLAEASVMTQL “+++” 228 FLGGLSPGV “+++” 229 FLLNLQNCHL “++++” 230 FLQDSKVIFV “+++” 231 FLYIRQLAI “+++” 232 FMHQIIDQV “++++” 233 GIIDINVRL “++” 234 GLDDAEYAL “++” 235 GLDDLLLFL “+++” 236 GLLESGRHYL “+++” 237 GLQENLDVVV “++++” 238 GLVETELQL “+++” 239 ILAGEMLSV “+++” 240 ILARDILEI “+++” 241 ILGDILLKV “+++” 242 ILLGIQELL “+++” 243 ILPTLEKELFL “+++” 244 ILQALAVHL “+++” 245 KIMDYSLLLGV “++++” 246 KLDETGVAL “++” 247 KLKDRLPSI “++” 248 KTVEPPISQV “++” 249 LLPTGVFQV “+++” 250 LLVQEPDGLMV “+++” 251 LLYDNVPGA “+++” 252 NLLDPGSSYLL “+++” 253 NLWSVDGEVTV “+++” 254 QLIPKLIFL “+++” 255 YLFEEAISM “+++” 256 QLLPVSNVVSV “+++” 258 RLDYITAEI “+++” 259 RLLDEQFAVL “+++” 260 SLDDVEGMSV “+++” 261 SLVEAQGWLV “+++” 262 SLWNAGTSV “+++” 263 SQWEDIHVV “+++” 264 TILDYINVV “+++” 265 TLLADDLEIKL “+++” 266 TLLDQLDTQL “++” 267 TLLDWQDSL “+++” 268 TLLQVFHLL “+++” 269 TLTDEQFLV “++” 270 TVLPVPPLSV “+++” 271 VIRNIVEAA “++” 272 VLDELPPLI “+++” 273 VLGEYSYLL “+++” 274 VLLEYHIAYL “+++” 275 VLLFIEHSV “+++” 276 VLNDGAPNV “+++” 277 VMILKLPFL “++” 278 YLDDLLPKL “+++” 279 YMAPEVVEA “+++” 280 YTLDSLYWSV “+++” 281 NLLDDRGMTAL “+++” 282 LLRDGIELV “+++” 283 ILQPMDIHV “+++” 284 LLSAAEPVPA “++” 285 GVATAGCVNEV “++” 286 FLLEDLSQKL “+++” 287 FLWEEKFNSL “++++” 288 GLAESTGLLAV “+++” 289 ILEEQPMDMLL “+++” 291 ILLNEDDLVTI “+++” 292 AAALIIHHV “+++” 293 ALDIMIPMV “+++” 294 ALLDQLHTLL “++++” 295 ALLQKLQQL “+++” 296 FIAPTGHSL “++” 297 FLVEPQEDTRL “+++” 298 IILPVEVEV “+++” 299 ILEENIPVL “+++” 300 ILLNPAYDVYL “+++” 301 AASPIITLV “+++” 302 ILQDLTFVHL “+++” 303 ILSQPTPSL “+++” 304 LAIVPVNTL “+” 305 LLFPQIEGIKI “+++” 306 VVAEELENV “++” 307 LLLTKPTEA “+++” 308 SLYDVSRMYV “++++” 309 ILYGTQFVL “+++” 310 LLSTLHLLV “+++” 311 LLVDVEPKV “+++” 312 LLYNSTDPTL “+++” 313 LMADLEGLHL “+++” 314 LMKDCEAEV “+++” 315 LVYEAPETV “+++” 316 MLLEHGITL “+++” 317 NLLAHIWAL “+++” 318 NLQVTQPTV “+++” 319 QVIPQLQTV “+++” 320 TIAPVTVAV “++++” 321 RLLEFELAQL “+++” 322 SLASIHVPL “+++” 323 SLDLFNCEV “++++” 324 SLYSALQQA “+++” 325 TLENGVPCV “+++” 326 VLAFLVHEL “++++” 327 VLIKWFPEV “++++” 328 VLLPQETAEIHL “++++” 329 VLMDGSVKL “++++” 330 VLMWEIYSL “+++” 331 VLWELAHLPTL “+++” 332 VMIQHVENL “+++” 333 VTLEFPQLIRV “+++” 334 YLLEEKIASL “+++” 335 YLYQEQYFI “+++” 336 YMAVTTQEV “++++” 337 YMYEKESEL “++” 338 FLDMTNWNL “++++” 339 GLWGTVVNI “+++” 340 KLLEEICNL “++++” 341 LLAELPASVHA “+++” 342 SLITPLQAV “+++” 344 VLAFENPQV “+++” 345 YLIEPDVELQRI “+++” 346 VLVQVSPSL “+++” 347 YLGPVSPSL “+++” 348 ALAKPPVVSV “+++” 349 ALATHILSL “+++” 350 ALEDRVWEL “+++” 351 ALSEKLARL “+++” 352 ALVFELHYV “+++” 353 ATPMPTPSV “++” 354 FIMDDPAGNSYL “+++” 355 FIWPMLIHI “+++” 356 FLHDHQAEL “++” 357 FLIQEIKTL “+++” 358 FLTDYLNDL “+++” 359 FMQDPMEVFV “+++” 360 HLIDTNKIQL “+++” 361 ILQEFESKL “+++” 362 ILTELGGFEV “+++” 363 ITTEVVNELYV “+++” 364 KMDWIFHTI “+++” 365 LISPLLLPV “+++” 367 NLWSLVAKV “+++” 369 RLLDLENSLLGL “+++” 370 SIFASPESV “+++” 371 SLADDSVLERL “+++” 372 SLFGPLPGPGPALV “+++” 373 TLLADQGEIRV “++” 374 VLSVITEEL “+++” 375 VLWFKPVEL “+++” 376 VLYNQRVEEI “+++” 377 VVDGTCVAV “+++” 378 YILGKFFAL “+++” 379 YLAELVTPIL “+++” 380 YLDRKLLTL “+++” 381 YLLEENKIKL “+++” 382 YLLPLLQRL “++++” 383 YLLREWVNL “++++” 384 YMIGSEVGNYL “++++” 385 YTIPLAIKL “+++”

Example 6

Absolute Quantitation of Tumor Associated Peptides Presented on the Cell Surface.

The generation of binders, such as antibodies and/or TCRs, is a laborious process, which may be conducted only for a number of selected targets. In the case of tumor-associated and -specific peptides, selection criteria include but are not restricted to exclusiveness of presentation and the density of peptide presented on the cell surface. In addition to the isolation and relative quantitation of peptides as described in EXAMPLE 1, the inventors did analyze absolute peptide copies per cell as described in patent x. The quantitation of TUMAP copies per cell in solid tumor samples requires the absolute quantitation of the isolated TUMAP, the efficiency of TUMAP isolation, and the cell count of the tissue sample analyzed. An overview on our experimental approach is described below.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, a calibration curve was generated for each peptide using the internal standard method. The internal standard is a double-isotope-labelled variant of each peptide, i.e. two isotope-labelled amino acids were included in TUMAP synthesis. It differs from the tumor-associated peptide only in its mass but shows no difference in other physicochemical properties (Anderson et al., 2012). The internal standard was spiked to each MS sample and all MS signals were normalized to the MS signal of the internal standard to level out potential technical variances between MS experiments. The calibration curves were prepared in at least three different matrices, i.e. HLA peptide eluates from natural samples similar to the routine MS samples, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to the signal of the internal standard and a calibration curve was calculated by logistic regression. For the quantitation of tumor-associated peptides from tissue samples, the respective samples were also spiked with the internal standard; the MS signals were normalized to the internal standard and quantified using the peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins from tissue samples is associated with a certain loss of the protein of interest. To determine the efficiency of TUMAP isolation, peptide/MHC complexes were generated for all TUMAPs selected for absolute quantitation. To be able to discriminate the spiked from the natural peptide/MHC complexes, single-isotope-labelled versions of the TUMAPs were used, i.e. one isotope-labelled amino acid was included in TUMAP synthesis. These complexes were spiked into the freshly prepared tissue lysates, i.e. at the earliest possible point of the TUMAP isolation procedure, and then captured like the natural peptide/MHC complexes in the following affinity purification. Measuring the recovery of the single-labelled TUMAPs therefore allows conclusions regarding the efficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples and was comparable among these tissue samples. In contrast, the isolation efficiency differs between individual peptides. This suggests that the isolation efficiency, although determined in only a limited number of tissue samples, may be extrapolated to any other tissue preparation. However, it is necessary to analyze each TUMAP individually as the isolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected to absolute peptide quantitation, the inventors applied DNA content analysis. This method is applicable to a wide range of samples of different origin and, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, a tissue sample is processed to a homogenous lysate, from which a small lysate aliquot is taken. The aliquot is divided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content from each DNA isolation is quantified using a fluorescence-based DNA quantitation assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at least two replicates.

In order to calculate the cell number, a DNA standard curve from aliquots of single healthy blood cells, with a range of defined cell numbers, has been generated. The standard curve is used to calculate the total cell content from the total DNA content from each DNA isolation. The mean total cell count of the tissue sample used for peptide isolation is extrapolated considering the known volume of the lysate aliquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculated the number of TUMAP copies per cell by dividing the total peptide amount by the total cell count of the sample, followed by division through isolation efficiency. Copy cell number for selected peptides are shown in Table 12.

TABLE 12 Absolute copy numbers. The table lists the results of absolute peptide quantitation in NSCLC tumor samples. The median number of copies per cell are indicated for each peptide: <100 = +; > = 100 = ++; > = 1,000 +++; > = 10,000 = ++++. The number of samples, in which evaluable, high quality MS data are available, is indicated. Number of quantifiable Seq ID Sequence Copy Number Category samples 62 GIIDGSPRL + 17 153 SLLAELHVL ++ 17

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The invention claimed is:
 1. A method of eliciting an immune response in a patient who has cancer, comprising administering to the patient a composition comprising a population of activated T cells that selectively recognize the cancer cells that present a peptide consisting of the amino acid sequence of GIIDGSPRL (SEQ ID NO: 62), wherein said cancer is selected from the group consisting of glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell and small cell lung cancer, non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer, urinary bladder cancer, head and neck squamous cell carcinoma, and uterine cancer.
 2. The method of claim 1, wherein the activated T cells are produced by contacting in vitro T cells with the peptide loaded onto human class I MHC molecules expressed on the surface of an antigen-presenting cell for a period of time sufficient to activate said T cells.
 3. The method of claim 2, wherein the antigen presenting cell is infected with recombinant virus expressing the peptide.
 4. The method of claim 3, wherein the antigen presenting cell is a dendritic cell or a macrophage.
 5. The method of claim 1, wherein the T cells are autologous to the patient.
 6. The method of claim 1, wherein the T cells are obtained from a healthy donor.
 7. The method of claim 1, wherein the T cells are obtained from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.
 8. The method of claim 1, wherein the activated T cells are expanded in vitro.
 9. The method of claim 1, wherein the peptide is in a complex with a class I MHC molecule.
 10. The method of claim 1, wherein the population of activated T cells comprises CD8-positive cells.
 11. The method of claim 1, wherein the composition further comprises an adjuvant.
 12. The method of claim 11, wherein the adjuvant is selected from the group consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, Sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulation with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 13. The method of claim 1, wherein the immune response comprises a cytotoxic T cell response.
 14. The method of claim 1, wherein the cancer is chronic lymphocytic leukemia.
 15. The method of claim 1, wherein the cancer is acute myeloid leukemia.
 16. The method of claim 1, wherein the cancer is non-Hodgkin lymphoma.
 17. A method of eliciting an immune response in a patient who has glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell and small cell lung cancer, non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer, urinary bladder cancer, head and neck squamous cell carcinoma, and/or uterine cancer, comprising administering to said patient a composition comprising a peptide in the form of a pharmaceutically acceptable salt, wherein said peptide consists of the amino acid sequence of GIIDGSPRL (SEQ ID NO: 62), thereby inducing a T-cell response to the glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell and small cell lung cancer, non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer, urinary bladder cancer, head and neck squamous cell carcinoma, and/or uterine cancer, wherein the T cell response is a cytotoxic T cell response. 